VEGF is essential for the growth and migration of human hepatocellular carcinoma cells

Vascular endothelial growth factor (VEGF) plays a crucial role in tumor angiogenesis. VEGF induces new vessel formation and tumor growth by inducing mitogenesis and chemotaxis of normal endothelial cells and increasing vascular permeability. However, little is known about VEGF function in the proliferation, survival or migration of hepatocellular carcinoma cells (HCC). In the present study, we have found that VEGF receptors are expressed in HCC line BEL7402 and human HCC specimens. Importantly, VEGF receptor expression correlates with the development of the carcinoma. By using a comprehensive approaches including TUNEL assay, transwell and wound healing assays, migration and invasion assays, adhesion assay, western blot and quantitative RT-PCR, we have shown that knockdown of VEGF165 expression by shRNA inhibits the proliferation, migration, survival and adhesion ability of BEL7402. Knockdown of VEGF165 decreased the expression of NF-κB p65 and PKCα while increased the expression of p53 signaling molecules, suggesting that VEGF functions in HCC proliferation and migration are mediated by P65, PKCα and/or p53.     

A novel derivative of decursin, CSL-32, blocks migration and production of inflammatory mediators and modulates PI3K and NF-κB activities in HT1080 cells

Decursin and related coumarin compounds in herbal extracts have a number of biological activities against inflammation, angiogenesis and cancer. We have analysed a derivative of decursin (CSL-32) for activity against inflammatory activation of cancer cells, such as migration, invasion and expression of pro-inflammatory mediators. The human fibrosarcoma cell line, HT1080, was treated with TNFα (tumour necrosis factor α) in the presence or absence of CSL-32. The cellular responses and modification of signalling adapters were analysed with respect to the production of pro-inflammatory mediators, as also migration, adhesion and invasion. Treatment of HT1080 cells with CSL-32 inhibited their proliferation, without affecting cell viability, and TNFα-induced expression of pro-inflammatory mediators, such as MMP-9 (matrix metalloproteinase-9) and IL-8 (interleukin-8). CSL-32 also suppressed phosphorylation and degradation of IκB (inhibitory κB), phosphorylation of p65 subunit of NF-κB (nuclear factor-κB) and nuclear translocation of NF-κB, which are required for the expression of pro-inflammatory mediators. In addition, CSL-32 inhibited invasion and migration of HT1080 cells, as also cellular adhesion to fibronectin, an ECM (extracellular matrix) protein. CSL-32 treatment resulted in a dose-dependent inhibition of PI3K (phosphoinositide 3-kinase) activity, required for the cellular migration. The analyses show that CSL-32 inhibits processes associated with inflammation, such as the production of pro-inflammatory mediators, as well as adhesion, migration and invasion in HT1080 cells.     

Ginsenoside metabolite compound K differentially antagonizing tumor necrosis factor-α-induced monocyte-endothelial trafficking

Human leukocyte endothelial adhesion and transmigration occur in the early stage of the pathogenesis of atherosclerosis. Vascular endothelial cells are targeted by pro-inflammatory cytokines modulating many gene proteins responsible for cell adhesion, thrombosis and inflammatory responses. This study examined the potential of compound K to inhibit the pro-inflammatory cytokine TNF-α induction of monocyte adhesion onto TNF-α-activated human umbilical vein endothelial cells (HUVEC). HUVEC were cultured with 10 ng/ml TNF-α with individual ginsenosides of Rb1, Rc, Re, Rh1 and compound K (CK). Ginsenosides at doses of ?50 μM did not show any cytotoxicity. TNF-α induced THP-1 monocyte adhesion to HUVEC, and such induction was attenuated by Rh1 and CK. Consistently, CK suppressed TNF-α-induced expression of HUVEC adhesion molecules of VCAM-1, ICAM-1 and E-selectin, and also Rh1 showed a substantial inhibition. Rh1 and CK dampened induction of counter-receptors, α4/β1 integrin VLA-4 and αL/β2 integrin LFA-1 in TNF-α-treated THP-1 cells. Additionally, CK diminished THP-1 secretion of MMP-9 required during transmigration, inhibiting transendothelial migration of THP-1 cells. CK blunted TNF-α-promoted IL-8 secretion of HUVEC and CXCR1 expression of THP-1 monocytes. Furthermore, TNF-α-activated endothelial IκB phosphorylation and NF-κB nuclear translocation were disturbed by CK, and TNF-α induction of α4/β1 integrin was abrogated by the NF-κB inhibitor SN50. These results demonstrate that CK exerts anti-atherogenic activity with blocking leukocyte endothelial interaction and transmigration through negatively mediating NF-κB signaling.     

Human B cells become highly responsive to macrophage-inflammatory protein-3 alpha/CC chemokine ligand-20 after cellular activation without changes in CCR6 expression or ligand binding

CCR6 is the only known receptor for the chemokine macrophage-inflammatory protein (MIP)-3alpha/CC chemokine ligand (CCL)20. We have shown previously that CCR6 is expressed on peripheral blood B cells, but CCR6 activity on these cells is low in in vitro assays. We report that MIP-3alpha/CCL20-induced calcium flux and chemotaxis can be enhanced significantly on peripheral blood and tonsillar B cells after activation by cross-linking surface Ag receptors. Of particular interest is the fact that the enhanced activity on B cells was not associated with an increase in CCR6 expression as assessed by levels of receptor mRNA, surface staining, or MIP-3alpha/CCL20 binding sites, or by a change in the affinity of the receptor for ligand. These data convincingly demonstrate that responses to a chemokine can be regulated solely by changes in the downstream pathways for signal transduction resulting from Ag receptor activation, and establish CCR6 as an efficacious receptor on human B cells.     

Nobiletin, a citrus flavonoid, suppresses invasion and migration involving FAK/PI3K/Akt and small GTPase signals in human gastric adenocarcinoma AGS cells

Nobiletin, a compound isolated from citrus fruits, is a polymethoxylated flavone derivative shown to have anti-inflammatory, antitumor, and neuroprotective properties. This study has investigated that nobiletin exerted inhibitory effects on the cell adhesion, invasion, and migration abilities of a highly metastatic AGS cells under non-cytotoxic concentrations. Data also showed nobiletin could inhibit the activation of focal adhesion kinase (FAK) and phosphoinositide-3-kinase/Akt (PI3K/Akt) involved in the downregulation of the enzyme activities, protein expressions, messenger RNA levels of matrix metalloproteinase-2 (MMP-2), and matrix metalloproteinase-2 (MMP-9). Also, our data revealed that nobiletin inhibited FAK/PI3K/Akt with concurrent reduction in the protein expressions of Ras, c-Raf, Rac-1, Cdc42, and RhoA by western blotting, whereas the protein level of RhoB increased progressively. Otherwise, nobiletin-treated AGS cells showed tremendously decreased in the phosphorylation and degradation of inhibitor of kappaBα (IκBα), the nuclear level of NF-κB, and the binding ability of NF-κB to NF-κB response element. Furthermore, nobiletin significantly decreased the levels of phospho-Akt and MMP-2/9 in Akt1-cDNA-transfected cells concomitantly with a marked reduction in cell invasion and migration. These results suggest that nobiletin can reduce invasion and migration of AGS cells, and such a characteristic may be of great value in the development of a potential cancer therapy.     

microRNA-148a-3p enhances the effects of sevoflurane on hepatocellular carcinoma cell progression via ROCK1 repression

BackgroundSevoflurane (SEVO) inactivates the aggressiveness of hepatocellular carcinoma (HCC) cells by mediating microRNAs (miRNAs). Hence, we delved into the functional role of miR-148a-3p mediated by SEVO in HCC.MethodsLiver cells (L02) and HCC cells (HCCLM3 and Huh7) were exposed to SEVO to detect cell viability in HCC. HCCLM3 and Huh7 cells were treated with restored miR-148a-3p or depleted Rho-associated protein kinase 1 (ROCK1) to elucidate their roles in HCC cells' biological characteristics. HCCLM3 and Huh7 cells were treated with SEVO, and/or vectors that changed miR-148a-3p or ROCK1 expression to identify their combined functions in HCC cell progression. Tumor xenograft in nude mice was performed to determine growth ability of tumor. The target relationship between miR-148a-3p and ROCK1 was verified.ResultsSEVO inhibited proliferation, invasion and migration and enhanced apoptosis of HCCLM3 and Huh7 cells. MiR-148a-3p up-regulation or ROCK1 down-regulation inhibited HCCLM3 and Huh7 cell progression. ROCK1 was determined to be target gene of miR-148a-3p. Down-regulating miR-148a-3p or overexpressing ROCK1 mitigated cell aggressiveness inhibition caused by SEVO.ConclusionOur study elucidates that microRNA-148a-3p enhances the effects of sevoflurane on inhibiting proliferation, invasion and migration and enhancing apoptosis of HCC cells through suppression of ROCK1.     

Ectopic expression of CC chemokine receptor 7 promotes prostate cancer cells metastasis via Notch1 signaling

There currently exists no satisfactory treatment for patients with prostate cancer with local evolution and distant metastasis. Previous studies have confirmed the importance of CC chemokine receptor 7 (CCR7) in the invasion and metastasis of prostate cancer. And increasing evidence prove that Notch1 can play diametrically opposite roles in the development and progression of different tumors. To demonstrate the correlation between CCR7 and Notch1, PC-3 cells were transfected with pcDNA3.1-CCR7 or CCR7 si-RNA, respectively. Then Western blot analysis was used to detect the expressions of Notch1, ERK, P38, JNK, NF-κB, MMP-9, and epithelial-mesenchymal transition (EMT)-related proteins. Moreover, matrigel invasion assays were performed to assess the migratory and invasive activities of PC-3 cells. PcDNA3.1-CCR7 increased the expression of Notch1, phospho-MAPK, phospho-P65, MMP-9, N-cadherin, and Snail in PC-3 cells, but decreased the expression of E-cadherin. PcDNA3.1-CCR7 also promoted the migration and invasion of PC-3 cells. However, CCR7 si-RNA reversed the effect of pcDNA3.1-CCR7 in PC-3 cells. And MAPK and NF-κB pathway inhibitors were used to testify that activation of Notch1 induces EMT through MAPK and NF-κB pathway. All these results indicate that upregulation of Notch1 by CCR7 can accelerate the evolution of EMT and develop the invasion and metastasis in prostate cancer cells by activating MAPK and NF-κB signaling pathways in prostate cancer cells, which provides a new molecular evidence for targeted therapy in metastatic prostate cancer.     

Time-Course Changes and New Expressions of MIP-3α and Its Receptor,CCR6, in the Gerbil Hippocampal CA1 Area Following Transient Global Cerebral Ischemia

Macrophage inflammatory protein-3α (MIP-3α) and its sole receptor, CCR6, play pivotal roles in neuroinflammatory processes induced by brain ischemic insults. In this study, we investigated transient ischemia-induced changes in MIP-3α and CCR6 protein expressions in the hippocampal CA1 area following 5 min of transient global cerebral ischemia (tgCI) in gerbils. Both MIP-3α and CCR6 immunoreactivities were very strongly expressed in pyramidal neurons of the CA1 area from 6 h to 1 day after tgCI and were hardly shown 4 days after tgCI. In addition, strong MIP-3α immunoreactivity was newly expressed in astrocytes 6 h after tgCI. These results indicate that tgCI causes apparent changes in MIP-3α and CCR6 expressions in pyramidal neurons and astrocytes in the hippocampal CA1 area and suggest that tgCI-induced changes in MIP-3α and CCR6 expressions might be closely associated with neuroinflammatory processes in brain ischemic regions.     

Syndecan-1 and syndecan-4 are involved in RANTES/CCL5-induced migration and invasion of human hepatoma cells

Background

We previously demonstrated that the CC-chemokine Regulated upon Activation, Normal T cell Expressed and Secreted (RANTES)/CCL5 exerts pro-tumoral effects on human hepatoma Huh7 cells through its G protein-coupled receptor, CCR1. Glycosaminoglycans play major roles in these biological events.

Methods

In the present study, we explored 1/ the signalling pathways underlying RANTES/CCL5-mediated hepatoma cell migration or invasion by the use of specific pharmacological inhibitors, 2/ the role of RANTES/CCL5 oligomerization in these effects by using a dimeric RANTES/CCL5, 3/ the possible involvement of two membrane heparan sulfate proteoglycans, syndecan-1 (SDC-1) and syndecan-4 (SDC-4) in RANTES/CCL5-induced cell chemotaxis and spreading by pre-incubating cells with specific antibodies or by reducing SDC-1 or -4 expression by RNA interference.

Results and conclusion

The present data suggest that focal adhesion kinase phosphorylation, phosphoinositide 3-kinase-, mitogen-activated protein kinase- and Rho kinase activations are involved in RANTES/CCL5 pro-tumoral effects on Huh7 cells. Interference with oligomerization of the chemokine reduced RANTES/CCL5-mediated cell chemotaxis. This study also indicates that SDC-1 and -4 may be required for HepG2, Hep3B and Huh7 human hepatoma cell migration, invasion or spreading induced by the chemokine. These results also further demonstrate the involvement of glycosaminoglycans as the glycosaminoglycan-binding deficient RANTES/CCL5 variant, in which arginine 47 was replaced by lysine, was devoid of effect.

General significance

The modulation of RANTES/CCL5-mediated cellular effects by targeting the chemokine-syndecan interaction could represent a new therapeutic approach for hepatocellular carcinoma.     

Silencing of PTGS2 exerts promoting effects on angiogenesis endothelial progenitor cells in mice with ischemic stroke via repression of the NF-κB signaling pathway

The objective of the current study is to investigate the effect of PTGS2 on proliferation, migration, angiogenesis and apoptosis of endothelial progenitor cells (EPCs) in mice with ischemic stroke through the NF-κB signaling pathway. Middle cerebral artery occlusion (MCAO) model was established in mice. EPCs were identified, in which ectopic expression and depletion experiments were conducted. The mRNA and protein expression of related factors in tissues and cells were measured. Besides, proliferation, migration, angiogenesis, and apoptosis, as well as cell cycle distribution, of cells were determined. MCAO mice showed overexpression of interleukin-6 (IL-6), IL-17, and IL-23, and increased positive protein expression of PTGS2, as well as expression of PTGS2, nuclear factor-κB (NF-κB), tumor suppressor region 1 (TSP-1) and Bcl-2-associated X protein (Bax), but underexpression of vascular endothelial growth factor (VEGF), S-phase kinase associated protein 2 (Skp2), and B-cell lymphoma 2 (Bcl-2). Moreover, ectopic expression of tumor necrosis factor-α significantly elevated the expression of PTGS2, NF-κB, TSP-1, and Bax, as well as cell apoptosis and cell cycle arrest, but decreased the expression of VEGF, Skp2, and Bcl-2, as well as proliferation, migration and angiogenesis of EPCs, and the PTGS2-siRNA group showed an opposite trend. Taken together, we conclude that the specific knockdown of PTGS2 expression could repress the NF-κB signaling pathway, thereby inhibits apoptosis and promotes proliferation, migration and angiogenesis of EPCs, providing protective effect on mice with ischemic stroke.     

Knockdown of MACC1 expression suppressed hepatocellular carcinoma cell migration and invasion and inhibited expression of MMP2 and MMP9

Expression of MACC1 (metastasis-associated in colon cancer-1) protein is associated with metastasis of various human cancers. This study analyzed MACC1 protein expression in hepatocellular carcinoma (HCC) tissue specimens and then investigated the effects of MACC1 knockdown on HCC cell migration and invasion, and gene expression levels. Sixty pairs of HCC and adjacent normal liver tissues from HCC patients were analyzed for MACC1 expression immunohistochemically. The HCC cell lines Hep3B, Huh7, MHCC97H, SMMC-7721, Bel-7402, and HepG2 and the normal liver cell line LO2 were used to assess expressions of MACC1 mRNA and MACC1 protein using qRT-PCR and western blot, respectively. MACC1 short hairpin RNA (shRNA) was used to knockdown MACC1 protein expression in Huh7 cells. Changes in the tumor phenotype of these cells were analyzed with wound healing assay and invasion assays, and differences in gene expression were evaluated via western blot. Immunofluorescence was used to locate MACC1 protein in the above cell lines. MACC1 was highly expressed in HCC tissues and the nuclear expression of MACC1 protein was associated with poor tumor differentiation and intrahepatic metastasis or portal invasion. Moreover, MACC1 mRNA and MACC1 protein was also expressed in HCC cell lines. Immunostaining showed that MACC1 protein was localized in both nuclei and cytoplasm of HCC cell lines and the nuclear localization of MACC1 protein was associated with increased aggressiveness of HCC in cell lines. Knockdown of MACC1 expression using MACC1-shRNA reduced Huh7 cell migration and invasion abilities, which was associated with downregulation of MMP2, MMP9, and c-Met proteins in Huh7 cells. Localization of MACC1 protein to the nucleus may predict HCC progression. Knockdown of MACC1 expression using MACC1 shRNA warrants further evaluation as a novel therapeutic strategy for control of HCC.     

Recruitment of human cord blood-derived endothelial colony-forming cells to sites of tumor angiogenesis

Background aimsEndothelial progenitor cells (EPCs) specifically home to sites of malignant growth, rendering them attractive for anti-cancer therapies. Data are conflicting on the phenotype and quantitative contribution toward tumor angiogenesis based on differing culture assays to outgrow EPCs. To evaluate the origin and early phenotype of EPCs and to define a population with enhanced tumor-targeting capacity, we evaluated a hierarchy of cord blood-derived EPCs modeling the multi-step nature of tumor homing.MethodsCD34⁺ mononuclear cells were isolated from fresh cord blood and cultured to derive endothelial colony-forming cells (ECFCs). Human umbilical vein endothelial cells (HUVECs) served as control. Using intra-vital microscopy, the recruitment was analyzed in mice bearing C6 xenografts. Adhesion, migration, transmigration and differentiation were further addressed.ResultsWithin the primary passage, ECFCs underwent a rapid maturation from a CD45⁺ and CD31⁺ phenotype to a CD45^? and endothelial marker positive phenotype. Assessing in vivo tumor recruitment, ECFCs had the highest activity in all steps analyzed. In vitro, ECFCs demonstrated significantly higher adhesion under static and flow conditions. Similarly, ECFCs exhibited highest migratory and trans-migratory activity toward tumor-conditioned medium. On subcutaneous implantation, only ECFCs formed blood vessels covered with perivascular cells, similar to HUVECs.ConclusionsOur study indicates that ECFCs emerge from a CD45⁺ and CD31⁺ progenitor and rapidly mature in culture. ECFCs have a significantly higher potential for tumor targeting than non-cultured CD34⁺ cells and HUVECs. They are ideal candidates for future cell-based anti-cancer therapies.     

Poly (ADP-ribose) polymerase inhibition synergizes with the NF-κB inhibitor DHMEQ to kill hepatocellular carcinoma cells

Poly (ADP-ribose) polymerase (PARP) enzymes play a key role in the cellular machinery responsible for DNA repair. Dehydroxymethylepoxyquinomicin (DHMEQ), a new inhibitor of NF-κB, induces oxidative stress and DNA damage. The effects of DHMEQ in combination with Olaparib (PARP inhibitor) were studied on hepatocellular carcinoma (HCC) cells. The DHMEQ–Olaparib combination synergistically inhibited cell viability, cell proliferation and colony formation of Hep3B, but had additive effects on Huh7 cells. The synergistic effects of the combination correlated with increased apoptosis, caspase 3/7 activity and PARP cleavage. There was an induction of an endoplasmic reticulum (ER) stress response with significant up-regulation of CHOP and TRB3 genes and splicing of XBP1 mRNA in Hep3B cells but not in Huh7 cells. Silencing of the TRB3 mRNA in Hep3B cells reversed the reduction in viability caused by DHMEQ–Olaparib treatment, while depletion of unspliced XBP1 mRNA in DHMEQ–Olaparib-treated Huh7 cells reduced viability. ROS production was increased after DHMEQ–Olaparib treatment of Hep3B, which caused DNA damage by an accumulation of γH2AX, increased AKT phosphorylation and reduced cell viability. The combination reduced Rad51 nuclear foci in Hep3B cells (not Huh7 cells), and silencing of Rad51 enhanced sensitivity of Huh7 cells to the DHMEQ–Olaparib combination. Knockdown of AKT in Hep3B cells restored the number of Rad51 nuclear foci after DHMEQ–Olaparib treatment. In summary, the DHMEQ–Olaparib combination induced ROS production, which killed HCC cells via DNA damage that could not be repaired by Rad51.SummaryPARPs and NF-κB are frequently deregulated in HCC. The DHMEQ–Olaparib combination exerted synergistic anti-tumour effects on HCC cells through ROS production via DNA damage that could not be repaired by Rad51. This suggested that the DHMEQ–Olaparib combination could be used to treat tumours that were resistant to Olaparib treatment.     

Extracellular nucleotide inhibits cell proliferation and negatively regulates Toll-like receptor 4 signalling in human progenitor endothelial cells

Extracellular nucleotides mediate a wide range of physiological effects by interacting with plasma membrane P2 purinergic receptors. P2 receptors are expressed in certain kinds of stem cells, and function to induce cytokine expression and to modulate cell proliferation. We have analysed the expression and the function of P2 receptors in human umbilical cord blood-derived EPCs (endothelial progenitor cells). EPCs expressed P2X4,6,7 and P2Y2,4,11,13,14 receptors and extracellular ATP inhibited EPCs proliferation. As in a previous study, EPCs expressed functional TLR4 (Toll-like receptor 4) and activation of TLR4 by LPS (lipopolysaccharide) evoked a pro-inflammatory immune response. When human EPCs were stimulated with LPS and nucleotides, ATP or UTP inhibited the expression of pro-inflammatory cytokines including MCP-1 (monocyte chemoattractant protein-1), IFNα (interferon α), TNFα (tumour necrosis factor α) and adhesion molecule VCAM-1 (vascular cell adhesion molecule 1) induced by LPS. ATP and UTP also down-regulated the gene expression of TLR4, CD14 and MyD88 (myeloid differentiation factor 88), a TLR adaptor molecule, and protein expression of CD14 and MyD88. Moreover, the phosphorylation of NF-κB (nuclear factor κB) p65 induced by TLR4 activation was inhibited partly by ATP or UTP at concentrations of 1-5 μM. These results suggest that extracellular nucleotides negatively regulate EPCs proliferation and TLR4 signalling.     

PKC�� take part in CCR7/NF-��B autocrine signaling loop in CCR7-positive squamous cell carcinoma of head and neck

Metastatic squamous cell carcinoma of head and neck (SCCHN) has been shown to express chemokine receptor 7 (CCR7). The role of nuclear factor-κB (NF-κB) in propagating an autocrine signaling loop in CCR7-positive SCCHN cells may provide a clinically useful biomarker of disease status and response to therapy. In this article, we hypothesized that PKCα might be involved in the CCR7/NF-κB autocrine signaling loop. Results showed that CCL19 induced the activation of PKCα, and the increased activity of PKCα was abolished by CCR7 mAb. PKCα inhibition with G?6976 led to significant reduction in the activation and nuclear translocation of NF-κB induced by CCL19. Immunohistochemical assay also showed that CCR7 and PKCα were highly expressed in SCCHN and correlated with each other, which was significantly related to lymph node metastasis and clinical stage. Taken together, PKCα is involved in the CCR7/NF-κB autocrine signaling loop.     

Molecular characterization of CCR6: Involvement of multiple domains in ligand binding and receptor signaling

The CC chemokine receptor 6 (CCR6) is selectively expressed on memory T cells, B cells, and dendritic cells and appears to be involved in the initiation of a memory immune response. The only chemokine ligand for CCR6 is CCL20/MIP-3. In the present study, we attempted to define the extracellular domains (ECDs) of CCR6 responsible for CCL20/MIP-3 binding using a domain-swapping approach in which the ECDs of CCR6 were substituted with the corresponding CCR5 domains to generate various CCR6/CCR5 chimeras. These chimeras were tested for receptor expression, ligand binding, and functional activity as evaluated by calcium flux and chemotaxis. All chimeras showed respectable surface expression; however only one, substituted with extracellular loop 1 from CCR5, showed reduced functional activity. The general failure of functionality of the CCR6/CCR5 chimeras may imply that characteristics of each ECD are critical for coordination among all the ECDs of CCR6. Additionally, of interest, a chimera containing all of the ECDs from CCR5 in the context of CCR6 neither responded to CCR5 ligands nor served as a coreceptor for macrophage-tropic HIV-1. These results suggest that not only ECDs but also transmembrane and intracellular domains of CCR5 are involved in both ligand binding and coreceptor activity.