HBP induces the expression of monocyte chemoattractant protein-1 via the FAK/PI3K/AKT and p38 MAPK/NF-κB pathways in vascular endothelial cells

Inflammation is characterized by early influx of polymorphonuclear neutrophils (PMNs), followed by a second wave of monocyte recruitment. PMNs mediate monocyte recruitment via their release of heparin binding protein (HBP), which activates CCR2 (CC-chemokine receptor 2) on monocytes. However, the pathways for such signal transmission remain unknown. Accumulating evidences have highlighted the importance of leukocyte-endothelial cell interactions in the initiation of inflammation. In this study, an interesting finding is that HBP enhances the secretion of monocyte chemotactic protein 1(MCP-1), ligand of CCR2, from a third party, the endothelial cells (ECs). HBP-induced increase in MCP-1 production was demonstrated at the protein, mRNA and secretion levels. Exposure of ECs to HBP elicited rapid phosphorylation of FAK/PI3K/AKT and p38 MAPK/NF-κB signaling. MCP-1 levels were attenuated during the response to HBP stimulation by pretreatment with a FAK inhibitor (or siRNA), a PI3K inhibitor, an AKT inhibitor, a p38 inhibitor (or siRNA) and two NF-κB inhibitors. Additionally, pretreatment with inhibitors to FAK, PI3K and AKT led to a decrease in HBP-induced phosphorylation of p38/NF-κB axis. These results showed that HBP induced MCP-1 expression via a sequential activation of the FAK/PI3K/AKT pathway and p38 MAPK/NF-κB axis. Interestingly, the patterns of HBP regulation of the expression of the adhesion molecular VCAM-1 were similar to those seen in MCP-1 after pretreatment with inhibitors (or not). These findings may help to determine key pharmacological points of intervention, thus slowing the progress of inflammatory-mediated responses in certain diseases where inflammation is detrimental to the host.     

Hepatitis B X protein upregulates decoy receptor 3 expression via the PI3K/NF-κB pathway

Chronic hepatitis B (CHB) is associated with the development of hepatocellular carcinoma (HCC). Decoy receptor 3 (DcR3) is a tumor necrosis factor receptor that promotes tumor cell survival by inhibiting apoptosis and interfering with immune surveillance. Previous studies showed that DcR3 was overexpressed in HCC cells and that short hairpin RNA (shDcR3) sensitizes TRAIL-resistant HCC cells. However, the expression of DcR3 during hepatitis B virus (HBV) infection has not been investigated. Here, we demonstrated that DcR3 was overexpressed in CHB patients and that DcR3 upregulation was positively correlated with the HBV DNA load and liver injury (determined by histological activity index, serum alanine aminotransferase level, and aspartate aminotransferase level). We found that hepatitis B virus X protein (HBx) upregulated DcR3 expression in a dose-dependent manner, but this increase was blocked by NF-κB inhibitors. HBx also induced the activation of NF-κB, and the NF-κB subunits p65 and p50 upregulated DcR3 by directly binding to the DcR3 promoters. Inhibition of PI3K significantly downregulated DcR3 and inhibited the binding of NF-κB to the DcR3 promoters. Our results demonstrate that the HBx induced DcR3 expression via the PI3K/NF-κB pathway; this process may contribute to the development of HBV-mediated HCC.     

Sulforaphane controls TPA-induced MMP-9 expression through the NF-κB signaling pathway,but not AP-1, in MCF-7 breast cancer cells

Sulforaphane [1-isothiocyanato-4-(methylsulfinyl)-butane] is an isothiocyanate found in some cruciferous vegetables, especially broccoli. Sulforaphane has been shown to display anti-cancer properties against various cancer cell lines. Matrix metalloproteinase-9 (MMP-9), which degrades the extracellular matrix (ECM), plays an important role in cancer cell invasion. In this study, we investigated the effect of sulforaphane on 12-O-tetradecanoyl phorbol-13-acetate (TPA)-induced MMP-9 expression and cell invasion in MCF-7 cells. TPA-induced MMP-9 expression and cell invasion were decreased by sulforaphane treatment. TPA substantially increased NF-κB and AP-1 DNA binding activity. Pre-treatment with sulforaphane inhibited TPA-stimulated NF-κB binding activity, but not AP-1 binding activity. In addition, we found that sulforaphane suppressed NF-κB activation, by inhibiting phosphorylation of IκB in TPA-treated MCF-7 cells. In this study, we demonstrated that the inhibition of TPA-induced MMP-9 expression and cell invasion by sulforaphane was mediated by the suppression of the NF-κB pathway in MCF-7 cells. [BMB Reports 2013; 46(4): 201-206]     

Targeting MAPKs,NF-κB,and PI3K/AKT pathways by methyl palmitate ameliorates ethanol-induced gastric mucosal injury in rats

Excessive drinking of alcohol has been frequently associated with gastric injury; however, its underlying molecular mechanisms have been inadequately investigated. Methyl palmitate (MP) has demonstrated marked hepato-, cardio- and pulmonary protective features; however, its effects on ethanol-induced gastric injury have not been studied. The aim of the present study was to evaluate the potential gastroprotective activity of MP against ethanol-evoked gastric mucosal damage in rats and associated molecular mechanisms, for example, mitogen-activated protein kinases (MAPKs), nuclear factor κB (NF-κB), and phosphoinositide 3 kinase/protein kinase B (PI3K/AKT) pathways. The rat stomachs were examined in terms of the inflammatory, oxidative, and apoptotic perturbations. Current data demonstrated that pretreatment with MP attenuated the gross gastric damage, scores of ulcer index, area of mucosal lesions and histopathology outcomes; actions which were similar to the reference antiulcer omeprazole. MP inhibited NF-κB expression, its nuclear translocation, and the expression of its downstream signals, for example, tumor necrosis factor-α and myeloperoxidase besides restoration of interleukin-10 levels. Western blot analysis revealed that MP counteracted the disruption of MAPKs signaling via lowering p-c-Jun N-terminal kinase 1/2 (p-JNK1/2) expression and restoring the phospho-extracellular signal-regulated kinase 1/2 (p-ERK1/2) levels without affecting p-p38MAPK levels. Additionally, MP improved the antioxidant milieu via diminishing lipid peroxides and enhancing glutathione, glutathione peroxidase, total antioxidant capacity and mucosal nitric oxide. In the context of apoptosis, MP inhibited the cleavage of caspase-3 and poly(ADP-ribose)polymerase (PARP) and Bax protein expression with upregulating B cell lymphoma-2 expression (Bcl-2), thus, promoting gastric cellular survival. This was confirmed by MP activation of the PI3K/AKT pathway manifested by enhanced expression of PI3K p110α and p-AKT. Together, the present findings report the gastroprotective actions of MP mediated via its anti-inflammatory, antioxidant, and antiapoptotic actions. The underlying molecular mechanisms involve, at least partly, the modulation of MAPKs, NF-κB and PI3K/AKT transduction.     

NIK is involved in constitutive activation of the alternative NF-κB pathway and proliferation of pancreatic cancer cells

Pancreatic cancer has one of the poorest prognoses among human neoplasms. Constitutive activation of NF-κB is frequently observed in pancreatic cancer cells and is involved in their malignancy. However, little is known about the molecular mechanism of this constitutive NF-κB activation. Here, we show that the alternative pathway is constitutively activated and NF-κB-inducing kinase (NIK), a mediator of the alternative pathway, is significantly expressed in pancreatic cancer cells. siRNA-mediated silencing of NIK expression followed by subcellular fractionation revealed that NIK is constitutively involved in the processing of p100 and nuclear transport of p52 and RelB in pancreatic cancer cells. In addition, NIK silencing significantly suppressed proliferation of pancreatic cancer cells. These results clearly indicate that NIK is involved in the constitutive activation of the alternative pathway and controls cell proliferation in pancreatic cancer cells. Therefore, NIK might be a novel target for the treatment of pancreatic cancer.     

��-Actin is a downstream effector of the PI3K/AKT signaling pathway in myeloma cells

Interleukin 6 is the in vivo growth factor of myeloma cells. In response to IL-6 stimulation, the PI3K/AKT signaling pathway is activated in these cells. With comparative proteomic approaches, this study reveals many putative downstream effectors of the PI3K/AKT pathway. Mass spectrometry analysis of excised protein spots from 2-dimensional gel allowed the identification of proteins such as β-Actin, cyclophilin A, E3 SUMO-protein ligase PIAS-NY protein, HSP 27, PML, and transforming growth factor β-2. Among these putative effectors, β-Actin was chosen for further characterization. Phosphorylation of β-Actin by AKT upon IL-6 stimulation was confirmed by western blotting using a phospho-AKT substrate antibody. Interestingly, IL-6 significantly increased cell migration (P < 0.05) and the content of filamentous actin (P < 0.05). Therefore, IL-6 stimulation could have effects on the migration of myeloma cells, and the phosphorylation of β-Actin is probably involved in the process.     

CCR6 promotes tumor angiogenesis via the AKT/NF-κB/VEGF pathway in colorectal cancer

Chemokines and chemokine receptors play an important role in tumorigenesis. Angiogenesis is a vital part of the occurrence, development and metastasis of cancer. CCR6 is an important factor during tumor progression; however, its function in tumor angiogenesis is not fully understood. In our study, we found that CCR6 was significantly overexpressed in colorectal cancer (CRC) tissues and predicted a poor prognosis in CRC patients. We then verified the function of CCR6 on tumor angiogenesis in vivo and in vitro. We observed that silencing CCR6 could decrease angiogenesis by inhibiting the proliferation and migration of human umbilical vein endothelial cells (HUVECs), whereas overexpression of CCR6 can promote angiogenesis. Additionally, we investigated the molecular mechanisms and demonstrated that activation of the AKT/NF-κB pathway maybe involved in CCR6-mediated tumor angiogenesis, which was able to promote the secretion of vascular endothelial growth factor A (VEGF-A). In conclusion, CCR6 facilitates tumor angiogenesis via the AKT/NF-κB/VEGF pathway in colorectal cancer. CCR6 inhibition may be a novel option for anti-vascular treatment in CRC.     

Toll-like receptor 9 agonists up-regulates the expression of cyclooxygenase-2 via activation of NF-κB in prostate cancer cells

CpG-oligonucleotides (CpG-ODNs), mimicking bacterial DNA, have recently been shown to stimulate prostate cancer invasion in vitro via Toll-like receptor 9 (TLR9). Since cyclooxygenase 2 (COX-2), frequently overexpressed in multiple tumor types including prostate cancer, is a causal factor for tumor development, invasion and metastasis, an interesting question is raised whether TLR9 regulates COX-2 expression in prostate cancer cells. To address this question, herein we examined COX-2 expression in PC-3 cells stimulated with different doses and time courses of CpG-ODNs. The regulatory role of NF-κB in TLR9-mediated COX-2 expression was also investigated. CpG-ODN was found to up-regulate the expression of COX-2 in PC-3 cells in a dose- and time-dependent manner, but have little impact on COX-1 expression. Moreover, CpG-ODN also promoted nuclear translocation and activation of NF-κB, which appeared to be required for COX-2 induction by CpG-ODN. Overall, TLR9 up-regulates COX-2 expression in prostate cancer cells, at least partially through the activation of NF-κB, which may be implicated in tumor invasion and metastasis.     

Pathobiological implications of the expression of EGFR, pAkt, NF-κB and MIC-1 in prostate cancer stem cells and their progenies

The progression of prostate cancers (PCs) to locally invasive, androgen-independent and metastatic disease states is generally associated with treatment resistance and disease relapse. The present study was undertaken to establish the possibility of using a combination of specific oncogenic products, including epidermal growth factor receptor (EGFR), pAkt, nuclear factor-kappaB (NF-κB) and macrophage inhibitory cytokine-1 (MIC-1) as biomarkers and therapeutic targets for optimizing the management of patients with localized PC at earlier disease stages. The immunohistochemical and immunofluorescence data have revealed that the expression levels of EGFR, Ser(473)-pAkt, NF-κB p65 and MIC-1 proteins were significantly enhanced in the same subset of 76 cases of prostatic adenocarcinoma specimens during the disease progression and these biomarkers were expressed in a small subpopulation of CD133(+) PC cells and the bulk tumor mass of CD133(-) PC cells. Importantly, all of these biomarkers were also overexpressed in 80-100% of 30 PC metastasis bone tissue specimens. Moreover, the results have indicated that the EGF-EGFR signaling pathway can provide critical functions for the self-renewal of side population (SP) cells endowed with stem cell-like features from highly invasive WPE1-NB26 cells. Of therapeutic interest, the targeting of EGFR, pAkt, NF-κB or MIC-1 was also effective at suppressing the basal and EGF-promoted prostasphere formation by SP WPE1-NB26 cells, inducing disintegration of SP cell-derived prostaspheres and decreasing the viability of SP and non-SP WPE1-NB26 cell fractions. Also, the targeting of these oncogenic products induced the caspase-dependent apoptosis in chemoresistant SP WPE1-NB26 cells and enhanced their sensibility to the cytotoxic effects induced by docetaxel. These findings suggest that the combined use of EGFR, pAkt, NF-κB and/or MIC-1 may represent promising strategies for improving the accuracy of current diagnostic and prognostic methods and efficacy of treatments of PC patients in considering the disease heterogeneity, thereby preventing PC progression to metastatic and lethal disease states.     

Seminal plasma induces the expression of IL-1α in normal and neoplastic cervical cells via EP2/EGFR/PI3K/AKT pathway

Background

Cervical cancer is a chronic inflammatory disease of multifactorial etiology usually presenting in sexually active women. Exposure of neoplastic cervical epithelial cells to seminal plasma (SP) has been shown to promote the growth of cancer cells in vitro and tumors in vivo by inducing the expression of inflammatory mediators including pro-inflammatory cytokines. IL-1α is a pleotropic pro-inflammatory cytokine induced in several human cancers and has been associated with virulent tumor phenotype and poorer prognosis. Here we investigated the expression of IL-1α in cervical cancer, the role of SP in the regulation of IL-1α in neoplastic cervical epithelial cells and the molecular mechanism underlying this regulation.

Methods and results

Real-time quantitative RT-PCR confirmed the elevated expression of IL-1α mRNA in cervical squamous cell carcinoma and adenocarcinoma tissue explants, compared with normal cervix. Using immunohistochemistry, IL-1α was localized to the neoplastically transformed squamous, columnar and glandular epithelium in all cases of squamous cell carcinoma and adenocarcinomas explants studied. We found that SP induced the expression of IL-α in both normal and neoplastic cervical tissue explants. Employing HeLa (adenocarcinoma) cell line as a model system we identified PGE₂ and EGF as possible ligands responsible for SP-mediated induction of IL-1α in these neoplastic cells. In addition, we showed that SP activates EP2/EGFR/PI3kinase-Akt signaling to induce IL-1α mRNA and protein expression. Furthermore, we demonstrate that in normal cervical tissue explants the induction of IL-1α by SP is via the activation of EP2/EGFR/PI3 kinase-Akt signaling.

Conclusion

SP-mediated induction of IL-1α in normal and neoplastic cervical epithelial cells suggests that SP may promote cervical inflammation as well as progression of cervical cancer in sexually active women.

     

Cyr61 induces the expression of monocyte chemoattractant protein-1 via the integrin ανβ3, FAK,PI3K/Akt,and NF-κB pathways in retinal vascular endothelial cells

Diabetes causes a number of metabolic and physiological abnormalities in the retina. Many of the molecular and physiological abnormalities that develop during diabetic retinopathy are due to inflammation. Monocyte chemoattractant protein-1 (MCP-1) is an important factor involved in diabetic retinopathy. In a previous study, we found that cysteine-rich 61 (Cyr61), an important angiogenic factor, also plays an important role in diabetic retinopathy. In addition to the direct effects of Cyr61, we observed that Cyr61 can induce the expression of MCP-1. However, the mechanism through which this occurs is not completely understood in chorioretinal vascular endothelial cells. We therefore investigated the effects of Cyr61 on MCP-1 expression in this cell type. Cyr61 stimulated the expression of MCP-1 at the mRNA, protein, and secreted protein levels in a dose-dependent and time-dependent manner. Both total MCP-1 levels and secreted MCP-1 levels were attenuated during the response to Cyr61 stimulation by pretreatment with integrin ανβ3-blocking antibodies, a FAK inhibitor (PF573228), a PI3K inhibitor (LY294002), and an Akt inhibitor (A6730). Electrophoretic mobility shift assays revealed that the above inhibitors suppressed the activation of NF-κB. Additionally, deletion of the NF-κB-binding element in the MCP-1 gene promoter led to a decrease in expression in luciferase reporter assays. These results show that the induction of MCP-1 by Cyr61 is mediated through the activation of the integrin ανβ3, FAK, PI3K/Akt, and IKK/NF-κB pathways in chorioretinal vascular endothelial cells.     

Anti-inflammatory activities of inotilone from Phellinus linteus through the inhibition of MMP-9, NF-κB, and MAPK activation in vitro and in vivo

Inotilone was isolated from Phellinus linteus. The anti-inflammatory effects of inotilone were studied by using lipopolysaccharide (LPS)-stimulated mouse macrophage RAW264.7 cells and λ-carrageenan (Carr)-induced hind mouse paw edema model. Inotilone was tested for its ability to reduce nitric oxide (NO) production, and the inducible nitric oxide synthase (iNOS) expression. Inotilone was tested in the inhibitor of mitogen-activated protein kinase (MAPK)?[extracellular signal-regulated protein kinase (ERK), c-Jun NH(2)-terminal kinase (JNK), p38], and nuclear factor-κB (NF-κB), matrix-metalloproteinase (MMP)-9 protein expressions in LPS-stimulated RAW264.7 cells. When RAW264.7 macrophages were treated with inotilone together with LPS, a significant concentration-dependent inhibition of NO production was detected. Western blotting revealed that inotilone blocked the protein expression of iNOS, NF-κB, and MMP-9 in LPS-stimulated RAW264.7 macrophages, significantly. Inotilone also inhibited LPS-induced ERK, JNK, and p38 phosphorylation. In in vivo tests, inotilone decreased the paw edema at the 4(th) and the 5(th) h after Carr administration, and it increased the activities of catalase (CAT), superoxide dismutase (SOD), and glutathione peroxidase (GPx). We also demonstrated that inotilone significantly attenuated the malondialdehyde (MDA) level in the edema paw at the 5(th) h after Carr injection. Inotilone decreased the NO and tumor necrosis factor (TNF-α) levels on serum at the 5(th) h after Carr injection. Western blotting revealed that inotilone decreased Carr-induced iNOS, cyclooxygenase-2 (COX-2), NF-κB, and MMP-9 expressions at the 5(th) h in the edema paw. An intraperitoneal (i.p.) injection treatment with inotilone diminished neutrophil infiltration into sites of inflammation, as did indomethacin (Indo). The anti-inflammatory activities of inotilone might be related to decrease the levels of MDA, iNOS, COX-2, NF-κB, and MMP-9 and increase the activities of CAT, SOD, and GPx in the paw edema through the suppression of TNF-α and NO. This study presents the potential utilization of inotilone, as a lead for the development of anti-inflammatory drugs.