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.     

S-phase kinase-associated protein 2 promotes cell growth and motility in osteosarcoma cells

Skp2 (S-phase kinase-associated protein 2) plays an oncogenic role in a variety of human cancers. However, the function of Skp2 in osteosarcoma (OS) is elusive. Therefore, in the current study, we explore whether Skp2 exerts its oncogenic function in OS. The cell growth, apoptosis, invasion and cell cycle were measured in OS cells after Skp2 overexpression. We found that overexpression of Skp2 enhanced cell growth, and inhibited cell apoptosis in OS cells. Moreover, we observed that upregulation of Skp2 accelerated cell cycle progression in OS cells. Furthermore, the ability of migration and invasion was enhanced in Skp2 overexpressing OS cells. Mechanically, our Western blotting data suggested that Skp2 decreased the expression of E-cadherin, Foxo1, p21, and p57, but increased MMP-9 in OS cells. In conclusion, our study demonstrated that Skp2 exhibited an oncogenic function in OS cells, suggesting that inhibition of Skp2 may be a novel approach for the treatment of OS.     

Selective CCL5/RANTES-induced mast cell migration through interactions with chemokine receptors CCR1 and CCR4

Mast cells (MCs) accumulate at sites of allergic mucosal inflammation where they act as central effector and regulatory cells. Because chemokines are of vital importance in directing inflammatory leukocytes to the sites of inflammations, we have investigated the expression and function of CC-chemokine receptor (CCR) on human MCs. Two previously unrecognized MC-chemokine receptors, CCR1 and CCR4, could be identified on cord blood-derived MCs (CBMCs). CCR1 and CCR4 expressed on CBMCs exhibited a unique response profile. Of seven CCR1 and CCR4 agonists tested, only CCL5/RANTES act as an agonist inducing chemotaxis. The migration could be partially blocked by specific antibodies against CCR1 or CCR4, while a complete inhibition was achieved when both CCR1 and CCR4 were blocked. These results demonstrate that both CCR1 and CCR4 are functional receptors on human mast cells with capacity to mediate migration towards CCL5.     

A natural CCL5/RANTES variant antagonist for CCR1 and CCR3

The N-terminal domain of the chemokine CCL5/regulated upon activation normal T cell expressed and secreted (RANTES) has been shown to be critical for its biological activity on leukocytes. Several N-terminus-modified CCL5/RANTES derivatives, such as N-Terminal truncated CCL5/RANTES, Met-RANTES, and amino-oxypentane (AOP)-RANTES exhibited antagonist or partial agonist functions when investigated on the properties of their receptors CCR1, CCR3, and CCR5. Studying 95 African samples from Cameroon, we found a naturally occurring variant of CCL5/RANTES containing a missense mutation located in the first amino acid of the secreted form (S24F). S24F binds CCR1, CCR3, and CCR5 and triggers receptor down-modulation comparable to CCL5/RANTES. Moreover, in CCR5 positive cells, S24F elicits cellular calcium mobilization equivalent to that obtained with CCL5/RANTES. By contrast, S24F does not provoke any response in CCR1 and CCR3 positive cells. As CCL5/RANTES is able to attract different subtypes of leukocytes into inflamed tissue and intervenes in a wide range of allergic and autoimmune diseases, the discovery of this natural N-terminus-modified CCL5/RANTES analogue exhibiting differential effects on CCL5/RANTES receptors, opens up additional perspectives for therapeutic intervention.Nucleotide sequence data reported is available in the DDBJ/EMBL/GenBank databases under the accession number: DQ230537.     

CCL5/CCR5生物学轴在肿瘤中作用的研究进展

肿瘤因其易转移、易复发的特性成为一大难以治愈的疾病,已严重威胁到人类的生命健康。肿瘤微环境在肿瘤的生长、迁移、免疫逃逸、血管生成等过程中具有明显的促进作用。肿瘤微环境中细胞分泌的CCL5发挥的作用已受到越来越多的关注,且许多研究表明抑制CCL5/CCR5生物学轴可抑制肿瘤迁移、血管生成等,预示着这可能成为一个新的肿瘤治疗策略。本文总结了近年来关于CCL5/CCR5生物学轴的研究,包括CCL5/CCR5生物学轴介导的肿瘤生长迁移、血管生成、免疫逃逸等作用,及CCR5抑制剂在肿瘤治疗中的广阔前景。     

CCL21/CCR7 promotes G2/M phase progression via the ERK pathway in human non-small cell lung cancer cells

C-C chemokine receptor 7 (CCR7) contributes to the survival of certain cancer cell lines, but its role in the proliferation of human non-small cell lung cancer (NSCLC) cells remains vague. Proliferation assays performed on A549 and H460 NSCLC cells using Cell Counting Kit-8 indicated that activation of CCR7 by its specific ligand, exogenous chemokine ligand 21 (CCL21), was associated with a significant linear increase in cell proliferation with duration of exposure to CCL21. The CCL21/CCR7 interaction significantly increased the fraction of cells in the G(2)/M phase of the cell cycle as measured by flow cytometry. In contrast, CCL21/CCR7 had no significant influence on the G(0)/G(1) and S phases. Western blot and real-time PCR indicated that CCL21/CCR7 significantly upregulated expression of cyclin A, cyclin B1, and cyclin-dependent kinase 1 (CDK1), which are related to the G(2)/M phase transition. The expression of cyclin D1 and cyclin E, which are related to the G(0)/G(1) and G(1)/S transitions, was not altered. The CCL21/CCR7 interaction significantly enhanced phosphorylation of extracellular signal-regulated kinase (P-ERK) but not Akt, as measured by Western blot. LY294002, a selective inhibitor of PI3K that prevents activation of the downstream Akt, did not weaken the effect of CCL21/CCR7 on P-ERK. Coimmunoprecipitation further confirmed that there was an interaction between P-ERK and cyclin A, cyclin B1, or CDK1, particularly in the presence of CCL21. CCR7 small interfering RNA or PD98059, a selective inhibitor of MEK that disrupts the activation of downstream ERK, significantly abolished the effects of exogenous CCL21. These results suggest that CCL21/CCR7 contributes to the time-dependent proliferation of human NSCLC cells by upregulating cyclin A, cyclin B1, and CDK1 potentially via the ERK pathway.     

Human CC chemokine CCL23, a ligand for CCR1, induces endothelial cell migration and promotes angiogenesis

A number of chemokines induce angiogenesis and endothelial cells express several chemokine receptors. To date, only a limited number of CC chemokines for CCR1 have been reported to induce angiogenic responses. We investigated the ability of CCL23 (also known as MPIF-1, MIP-3, or CKbeta8) to promote angiogenesis, which induces chemotaxis of immune cells through CCR1. CCL23 promoted the chemotactic migration and differentiation of endothelial cells, and neovascularization in the chick chorioallantoic membrane. An N-terminal truncated form of CCL23 was at least 100-fold more potent than its intact form and was comparable to that of FGF in the angiogenic activities. Treatment with either pertussis toxin or anti-CCR1 antibody completely inhibited the CCL23-induced endothelial cell migration, indicating that endothelial cell migration was mediated through CCR1. CCL23 didn't promote the migration of HT1080 human fibrosarcoma cells that did not express CCR1. Our results suggest a role of CCL23 in angiogenesis in vitro as well as in vivo.     

IQGAP1 promotes cell motility and invasion

The dynamic processes of cell migration and invasion are largely coordinated by Rho family GTPases. The scaffolding protein IQGAP1 binds to Cdc42, increasing the amount of active Cdc42 both in vitro and in cells. Here we show that overexpression of IQGAP1 in mammalian cells enhances cell migration in a Cdc42- and Rac1-dependent manner. Importantly, cell motility was significantly decreased both by knock down of endogenous IQGAP1 using small interfering RNA and by transfection of a dominant negative IQGAP1 construct, IQGAP1DeltaGRD. Cell invasion was similarly altered by manipulating intracellular IQGAP1 concentrations. Moreover, invasion mediated by constitutively active Cdc42 was attenuated by IQGAP1DeltaGRD. Thus, IQGAP1 has a fundamental role in cell motility and invasion.     

Overexpression of integrin alphav promotes human osteosarcoma cell populated collagen lattice contraction and cell migration

Cells attach and interact with the extracellular matrix (ECM) through heterodimeric alphabeta integrin receptors. Specifically, the promiscuous alphavbeta3 integrin and the alpha2beta1 integrin receptors engage numerous matrix components to influence cell adhesion, cell motility, and matrix organization. However, the role of alphav integrin mediating cell-collagen interactions is not clear. In the in vitro cell populated collagen lattice (PCL), a model of cell-matrix interaction, integrin receptors play a role in lattice contraction. To elucidate alphav integrins' effects on cell-collagen interactions, human osteosarcoma (HOS) cells were transfected with alphav integrin (alphav-pcDNA 3.1+). Control HOS cells were transfected with pcDNA 3.1+ vector alone. HOS-alphav cell PCLs contracted to a greater degree than control HOS cell PCLs (P < or = 0.0001). RT-PCR revealed that HOS-alphav cells express both beta1 and beta3 integrins, indicating that alphav has the potential to form a partnership with either beta1 or beta3 integrin. The alphavbeta3 specific inhibitory antibody LM609 significantly retarded HOS-alphav cell PCL contraction (P < or = 0.001), suggesting that alphavbeta3 promotes enhanced HOS-alphav cell PCL contraction. When plated on plastic, control HOS cells show greater elongation compared to HOS-alphav cells. In addition, HOS-alphav cells migrated faster and to a greater degree than control HOS cells (P < or = 0.0001). The possibility that enhanced HOS-alphav cell migration and HOS-alphav cell PCL contraction was caused by increased myosin ATPase activity was examined. HOS-alphav cells showed less myosin ATPase activity than control HOS cells, by an ATP cell contraction bioassay. The enhancement of HOS-alphav cell migration and lattice contraction appears unrelated to increased myosin ATPase activity.     

PRRX1 promotes malignant properties in human osteosarcoma

Paired related homeobox 1 (PRRX1) is a marker of limb bud mesenchymal cells, and deficiency of p53 or Rb in Prrx1-positive cells induces osteosarcoma in several mouse models. However, the regulatory roles of PRRX1 in human osteosarcoma have not been defined. In this study, we performed PRRX1 immunostaining on 35 human osteosarcoma specimens to assess the correlation between PRRX1 level and overall survival. In patients with osteosarcoma, the expression level of PRRX1 positively correlated with poor prognosis or the ratio of lung metastasis. Additionally, we found PRRX1 expression on in 143B cells, a human osteosarcoma line with a high metastatic capacity. Downregulation of PRRX1 not only suppressed proliferation and invasion but also increased the sensitivity to cisplatin and doxorubicin. When 143B cells were subcutaneously transplanted into nude mice, PRRX1 knockdown decreased tumor sizes and rates of lung metastasis. Interestingly, forskolin, a chemical compound identified by Connectivity Map analysis using RNA expression signatures during PRRX1 knockdown, decreased tumor proliferation and cell migration to the same degree as PRRX1 knockdown. These results demonstrate that PRRX1 promotes tumor malignancy in human osteosarcoma.     

Fibronectin promotes rat Schwann cell growth and motility

Techniques are now available for culturing well characterized and purified Schwann cells. Therefore, we investigated the role of fibronectin in the adhesion, growth, and migration of cultured rat Schwann cells. Double-immunolabeling shows that, in primary cultures of rat sciatic nerve, Schwann cells (90%) rarely express fibronectin, whereas fibroblasts (10%) exhibit a granular cytoplasmic and fibrillar surface-associated fibronectin. Secondary cultures of purified Schwann cells do not express fibronectin. Exogenous fibronectin has a small effect on promoting the adhesion of Schwann cells to the substrate and does not significantly affect cell morphology, but it produced a surface fibrillar network on fibronectin on the secondary Schwann cells. Tritiated thymidine autoradiography revealed that addition of fibronectin to the medium, even at low concentrations, markedly stimulates Schwann cell proliferation, in both primary and secondary cultures. In addition, when cell migration was measured in a Boyden chamber assay, fibronectin was found to moderately, but clearly, stimulate directed migration or chemotaxis.     

Significantly increased CCL5/RANTES and CCR7 mRNA levels in localized scleroderma

Plaque-type morphea, the most common subtype of localized scleroderma (LS), is a connective tissue disease which is characterized by immunological dysregulation, vascular alterations, and skin fibrosis. We aimed to investigate the expression profiles of different cytokines and chemokines in patients with LS and healthy controls. Twenty patients with plaque-type morphea and 18 healthy controls were investigated. Skin biopsies were performed for real-time RT-PCR studies. Median mRNA of interleukin (IL)-6 was significantly higher expressed in LS than in normal skin. By contrast, median IL-1α mRNA levels were significantly decreased in LS as compared to controls. Median mRNA expression of CCL13, IL-1β, IL-2, and tumor necrosis factor-α did not significantly differ between LS lesions and healthy skin. However, we observed significantly increased median chemokine ligand 5/RANTES (CCL5/RANTES) and median chemokine receptor 7 (CCR7) mRNA expression in LS lesions as compared to healthy controls. CCL5/RANTES and CCR7 mRNA expression significantly correlated in LS lesions. We could confirm data of previous studies indicating that gene expression of IL-6 is up-regulated in LS lesions as compared to healthy skin. Moreover, we have shown for the first time a significant increase of mRNA levels of CCR7 and CCL5/RANTES in LS lesions indicating an important pathogenetic role of chemokines in LS.     

Ras activation mediates WISP-1-induced increases in cell motility and matrix metalloproteinase expression in human osteosarcoma

WISP-1 is a cysteine-rich protein that belongs to the CCN (Cyr61, CTGF, Nov) family of matrix cellular proteins. Osteosarcoma is a type of highly malignant tumor with a potent capacity to invade locally and cause distant metastasis. However, the effect of WISP-1 on migration activity in human osteosarcoma cells is mostly unknown. In this study, we first found that the expression of WISP-1 in osteosarcoma patients was significantly higher than that in normal bone and corrected with tumor stage. Exogenous treatment of osteosarcoma cells with WISP-1 promoted cell motility and matrix metalloproteinase (MMP)-2 and MMP-9 expression. In addition, the Ras and Raf-1 inhibitor or siRNA abolished WISP-1-induced cell migration and MMP expression. On the other hand, activation of the Ras, Raf-1, MEK, ERK, and NF-κB signaling pathway after WISP-1 treatment was demonstrated, and WISP-1-induced expression of MMPs and migration activity were inhibited by the specific inhibitor, and mutant of MEK, ERK, and NF-κB cascades. Taken together, our results indicated that WISP-1 enhances the migration of osteosarcoma cells by increasing MMP-2 and MMP-9 expression through the integrin receptor, Ras, Raf-1, MEK, ERK, and NF-κB signal transduction pathway.     

An abortive ligand-induced activation of CCR1-mediated downstream signaling event and a deficiency of CCR5 expression are associated with the hyporesponsiveness of human naive CD4+ T cells to CCL3 and CCL5

Human memory CD4(+) T cells respond better to inflammatory CCLs/CC chemokines, CCL3 and CCL5, than naive CD4(+) T cells. We analyzed the regulatory mechanism underlying this difference. Memory and naive CD4(+) T cells expressed similarly high levels of CCR1; however, CCR5 was only expressed in memory CD4(+) T cells at low levels. Experiments using mAbs to block chemokine receptors revealed that CCR1 functioned as a major receptor for the binding of CCL5 in memory and naive CD4(+) T cells as well as the ligand-induced chemotaxis in memory CD4(+) T cells. Stimulation of memory CD4(+) T cells with CCL5 activated protein tyrosine kinase-dependent cascades, which were significantly blocked by anti-CCR1 mAb, whereas this stimulation failed to induce these events in naive CD4(+) T cells. Intracellular expressions of regulator of G protein signaling 3 and 4 were only detected in naive CD4(+) T cells. Pretreatment of cell membrane fractions from memory and naive CD4(+) T cells with GTP-gamma S inhibited CCL5 binding, indicating the involvement of G proteins in the interaction of CCL5 and its receptor(s). In contrast, CCL5 enhanced the GTP binding to G(i alpha) and G(q alpha) in memory CD4(+) T cells, but not in naive CD4(+) T cells. Thus, a failure of the ligand-induced activation of CCR1-mediated downstream signaling event as well as a deficiency of CCR5 expression may be involved in the hyporesponsiveness of naive CD4(+) T cells to CCL3 and CCL5.