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1.
Lumican is a small leucine-rich proteoglycan (SLRP) present in the dermal extracellular matrix. Previous data from our laboratory demonstrated that lumican decreases melanoma progression in vivo. Here, we show that melanoma cell migration is decreased by lumican and that this effect is due to an enhanced cell adhesion. The adhesion of A375 human melanoma cells on lumican was dose-dependent and required Mg2+ and Mn2+ divalent cations. Using a panel of monoclonal antibodies directed against integrin subunits, we showed that A375 cells can bind to recombinant lumican through β1 type integrins. Moreover, the use of rhodocetin, an inhibitor of α2 integrin, suggested that this particular subunit might also be involved in the interaction with lumican. The increased β1 integrin-mediated adhesion of melanoma cells to lumican might explain, at least in part, the anti-invasive effect of this SLRP.  相似文献   

2.
Cell migration is a multistep process initiated by extracellular matrix components that leads to cytoskeletal changes and formation of different protrusive structures at the cell periphery. Lumican, a small extracellular matrix leucine-rich proteoglycan, has been shown to inhibit human melanoma cell migration by binding to α2β1 integrin and affecting actin cytoskeleton organization. The aim of this study was to determine the effect of lumican overexpression on the migration ability of human colon adenocarcinoma LS180 cells. The cells stably transfected with plasmid containing lumican cDNA were characterized by the increased chemotactic migration measured on Transwell filters. Lumican-overexpressing cells presented the elevated filamentous to monomeric actin ratio and gelsolin up-regulation. This was accompanied by a distinct cytoskeletal actin rearrangement and gelsolin subcellular relocation, as observed under laser scaning confocal microscope. Moreover, LS180 cells overexpressing lumican tend to form podosome-like structures as indicated by vinculin redistribution and its colocalization with gelsolin and actin at the submembrane region of the cells. In conclusion, the elevated level of lumican secretion to extracellular space leads to actin cytoskeletal remodeling followed by an increase in migration capacity of human colon LS180 cells. These data suggest that lumican expression and its presence in ECM has an impact on colon cancer cells motility and may modulate invasiveness of colon cancer.  相似文献   

3.
Lumican is a member of the small leucine-rich proteoglycan (SLRP) family. It contributes to the organisation of the collagen network and plays an important role in cell migration and tissue repair. The present study aimed to determine the influence of lumican expression on adhesion, anchorage-dependent and -independent growth, migration, in vitro invasion and in vivo melanoma growth. For that purpose, B16F1 mouse melanoma cells were stably transfected with an expression plasmid containing the complete lumican cDNA. Lumican expression by tumor cells did not change the proliferative activity of mouse melanoma cells in monolayer culture and did not influence either cell adhesion to extracellular matrix gel or type I collagen or cell spreading on these substrates. In contrast, lumican-transfected cells were characterized by a strong reduction of their anchorage-independent proliferation in agarose gel and capacity to invade extracellular matrix gel. After subcutaneous injections of transfected B16F1 cells in syngenic mice, lumican expression significantly decreased subcutaneous tumor formation in vivo, with a concomitant decrease of cyclin D1 expression. Lumican induced and/or increased the apoptosis of B16F1 cells. The results suggest that lumican is involved in the control of melanoma growth and invasion and may be considered, like decorin, as an anti-tumor factor from the extracellular matrix.  相似文献   

4.
Melanoma is a frequent and therapy-resistant human disease. Malignant melanocytes modulate their microenvironment in order to penetrate the dermal/epidermal junction and eventually invade the dermis. The small leucine-rich proteoglycans (SLRPs) constitute important constituents of the dermis extracellular matrix (ECM), participating in both the structural and the functional organization of the skin. The role of a keratan sulphate SLRP lumican, has recently been investigated in the growth and metastasis of several cancers. In this study, the expression of lumican was studied in two human melanoma cell lines (WM9, M5) as well as in normal neonatal human melanocytes (HEMN) using real time PCR, western blotting with antibodies against the protein core and keratan sulfate, and treatments with specific enzymes. Both human metastatic melanoma cell lines were found to express lumican mRNA and effectively secrete lumican in a proteoglycan form, characterized to be substituted mostly with keratan sulfate chains. Lumican mRNA was not detected in normal melanocytes. This is the first time that the synthesis and secretion of lumican in human melanoma cell lines is reported. The role of this proteoglycan in the development and progression of malignant melanoma has to be further investigated.  相似文献   

5.
Lumican, an extracellular matrix protein of the small leucine-rich proteoglycan family, has been shown to impede melanoma progression by inhibiting cell migration. In the present study, we show that lumican targets α2β1 integrin thereby inhibiting cell migration. A375 melanoma cells were transfected with siRNA directed against the α2 integrin subunit. Compared to A375 control cells, the anti-migratory effect of lumican was abrogated on transfected A375 cells. Moreover, lumican inhibited the chemotactic migration of Chinese hamster ovary (CHO) cells stably transfected with α2 integrin subunit (CHO-A2) but not that of wild-type CHO cells (CHO-WT) lacking this subunit. In contrast to CHO-WT cells, we observed in time-lapse microscopy a decrease of CHO-A2 cell migration speed in presence of lumican. Focal adhesion kinase phosphorylated at tyrosine-397 (pFAK) and total FAK were analysed in CHO-WT and CHO-A2 cells. A significant decrease of the ratio pFAK/FAK was shown in presence of recombinant human lumican. Using solid phase assays, a direct binding between lumican and the α2β1 integrin was demonstrated. This interaction did not involve the glycan moiety of lumican and was cation independent. Lumican was also able to bind the activated I domain of the α2 integrin subunit with a Kd ≥ 200 nM. In conclusion, we demonstrated for the first time that the inhibition of cell migration by lumican depends on a direct binding between the core protein of lumican and the α2β1 integrin.  相似文献   

6.
The consecutive steps of tumor growth, local invasion, intravasation, extravasation and invasion of anatomically distant sites are obligatorily perpetrated through specific interactions of the tumor cells with their microenvironment. Lumican, a class II small leucine-rich proteoglycans (SLRP) has been designated key roles both in extracellular matrix (ECM) organization and as an important modulator of biological functions. This review will critically discuss lumicans' roles in tumor development and progression. We will especially focus on correlating lumicans' expression and distribution in tumor tissues with: (1) the organization of the tumor matrices; (2) tumor cell signaling and functions; (3) tumor cell–matrix interface; (4) tumor angiogenesis; and (5) lumicans' potential roles in tumor-associated inflammatory response. Present knowledge of lumicans' biology provides a fundamental platform upon which to build and deepen our understanding of lumican function in tumorigenesis in order to be able to design credible anti-tumor approaches.  相似文献   

7.
AimsFormation of different protrusive structures by migrating cells is driven by actin polymerization at the plasma membrane region. Gelsolin is an actin binding protein controlling the length of actin filaments by its severing and capping activity. The main goal of this study was to determine the effect of gelsolin expression on the migration of human colon adenocarcinoma LS180 and melanoma A375 cells.Main methodsColon adenocarcinoma cell line LS180 was stably transfected with plasmid containing human cytoplasmic gelsolin cDNA tagged to enhanced green fluorescence protein (EGFP). Melanoma A375 cells were transfected with siRNAs directed against gelsolin. Real-time PCR and Western blotting were used to determine the level of gelsolin. The ability of actin to inhibit DNase I activity was used to quantify monomeric and total actin level and calculate the state of actin polymerization. Fluorescence confocal microscopy was applied to observe gelsolin and vinculin distribution along with actin cytoskeleton organization.Key findingsIncreased level of gelsolin expression leads to its accumulation at the submembranous region of the cell accompanied by distinct changes in the state of actin polymerization and an increase in the migration of LS180 cells. In addition, LS180 cells overexpressing gelsolin form podosome-like structures as indicated by vinculin redistribution and its colocalization with gelsolin and actin. Downregulation of gelsolin expression in melanoma A375 cells significantly reduces their migratory potential.SignificanceOur experimental data indicate that alterations in the expression level of gelsolin and its subcellular distribution may be directly responsible for determining migration capacity of human cancer cells.  相似文献   

8.
Lumican, a small leucine-rich proteoglycan of the extracellular matrix, presents potent anti-tumor properties. Previous works from our group showed that lumican inhibited melanoma cell migration and tumor growth in vitro and in vivo. Melanoma cells adhered to lumican, resulting in a remodeling of their actin cytoskeleton and preventing their migration. In addition, we identified a sequence of 17 amino acids within the lumican core protein, named lumcorin, which was able to inhibit cell chemotaxis and reproduce anti-migratory effect of lumican in vitro. The aim of the present study was to characterize the anti-tumor mechanism of action of lumcorin. Lumcorin significantly decreased the growth in monolayer and in soft agar of two melanoma cell lines – mice B16F1 and human SK-MEL-28 cells – in comparison to controls. Addition of lumcorin to serum free medium significantly inhibited spontaneous motility of these two melanoma cell lines. To characterize the mechanisms involved in the inhibition of cell migration by lumcorin, the status of the phosphorylation/dephosphorylation of proteins was examined. Inhibition of focal adhesion kinase phosphorylation was observed in presence of lumcorin. Since cancer cells have been shown to migrate and to invade by mechanisms that involve matrix metalloproteinases (MMPs), the expression and activity of MMPs were analyzed. Lumcorin induced an accumulation of an intermediate form of MMP-14 (~59kDa), and inhibited MMP-14 activity. Additionally, we identified a short, 10 amino acids peptide within lumcorin sequence, which was able to reproduce its anti-tumor effect on melanoma cells. This peptide may have potential pharmacological applications.  相似文献   

9.
Human osteosarcoma cell lines were recently shown to express and secrete the small leucine rich proteoglycan (SLRP) lumican, with the ability to regulate the growth and motility of these cells. In this study, lumican-deficient Saos 2 cells were demonstrated to have increased adhesive capability onto fibronectin (FN) (p≤0.01). Upon neutralization of endogenous transforming growth factor β2 (TGF-β2) activity, no difference in the ability of lumican siRNA-transfected and scramble siRNA-transfected Saos 2 cells to adhere onto FN was detected (p=NS). Exogenous TGF-β2 was shown to stimulate Saos 2 cell adhesion to FN (p≤0.01). These results therefore, suggest that the inverse correlation existing between lumican expression and Saos 2 cell adhesion is dependent on active TGF-β2 signaling. Furthermore, the significant increase in Smad 2 activation present in lumican-deficient cells (p≤0.01) was annulled in the presence of the anti-TGF-β2 peptide, demonstrating that lumican is an upstream regulator of the TGF-β2/Smad 2 signaling cascade. Crucial to FN-dependent adhesion, β1 integrin expression and pFAK activation were likewise identified as downstream TGF-β2 effectors regulated by lumican expression. In conclusion, this study demonstrates a novel out-in signaling circuit in human osteosarcoma cells: secreted to extracellular matrix lumican is an endogenous inhibitor of TGF-β2 activity, resulting in downstream effector modulation including pSmad 2, integrin β1 and pFAK to regulate osteosarcoma adhesion.  相似文献   

10.
In this report, we have investigated whether alterations of the morphological and functional aspects of the biosecretory membrane system are associated with the metastatic potential of tumor cells. To this end, we have analyzed the morphology of the Golgi complex, the cytoskeleton organization and membrane trafficking steps of the secretory pathway in two human melanoma A375 cell line variants with low (A375-P) and high metastatic (A375-MM) potential. Immunofluorescence analysis showed that in A375-P cells, the Golgi complex showed a collapsed morphology. Conversely, in A375-MM cells, the Golgi complex presented a reticular and extended morphology. At the ultrastructural level, the Golgi complex of A375-P cells was fragmented and cisternae were swollen. When the cytoskeleton was analyzed, the microtubular network appeared normal in both cell variants, whereas actin stress fibers were largely absent in A375-P, but not in A375-MM cells. In addition, the F-actin content in A375-P cells was significantly lower than in A375-MM cells. These morphological differences in A375-P cells were accompanied by acceleration and an increase in the endoplasmic reticulum to Golgi and the trans-Golgi network to cell surface membrane transport, respectively. Our results indicate that in human A375 melanoma cells, metastatic potential correlates with a well-structured morphofunctional organization of the Golgi complex and actin cytoskeleton.  相似文献   

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