Adhesion Of B16 Melanoma Cells Research Articles

Anti-metastatic effects of the sulfated polysaccharide ascophyllan isolated from Ascophyllum nodosum on B16 melanoma

We previously found that ascophyllan, a sulfated polysaccharide isolated from brown seaweed Ascophyllum nodosum, exhibited antitumor activity in sarcoma-180 tumor-bearing mice. In this study, we found that ascophyllan inhibited the migration and adhesion of B16 melanoma cells by reducing the expression of N-cadherin and enhancing the expression of E-cadherin in a concentration-dependent manner. Transwell invasion assay revealed that ascophyllan suppressed the invasion ability of B16 cells. It also inhibited the expression of matrix metalloprotease-9 (MMP-9) mRNA and the secretion of MMP-9 protein in B16 cells, a process that may involve the extracellular signal-regulated kinase (ERK) signaling pathway. Furthermore, ascophyllan administered intraperitoneally at 25 mg/kg showed anti-metastatic activity in a mouse model of metastasis induced by intravenous injection of B16 cells, and the number of lung surface metastatic nodules in ascophyllan-treated mice was significantly reduced compared to that in the untreated control mice. Since splenic natural killer cell activity enhanced in the mice injected with ascophyllan intraperitoneally, we suggest that ascophyllan may exhibit in vivo anti-metastatic activity on B16 melanoma cells through activation of the host immune system in addition to a direct action on cancer cells.

Fully phosphorothioate-modified CpG ODN with PolyG motif inhibits the adhesion of B16 melanoma cells in vitro and tumorigenesis in vivo.

Adhesion to the extracellular matrix and endothelial lining of blood vessels is critical for tumor cells to grow at original or metastatic sites. Inhibition of tumor cell adhesion can be an antitumor strategy. Guanosine-rich (G-rich) oligodeoxynucleotides (ODNs) can inhibit the adhesion of certain tumor cells. However, no data exist on how inclusion of the CpG motif in the G-rich sequence influences tumor cell adhesion and subsequent tumorigenesis. In this study, in vitro and in vivo assays were used to evaluate how a panel of ODN-containing contiguous guanosines and the CpG motif influenced adhesion of B16 melanoma cells. The results showed that a self-designed ODN, named BW001, containing the polyG motif and a full phosphorothioate modification backbone could inhibit B16 melanoma cell adhesion on a culture plate or on a plate coated with various substances. In vivo data revealed that B16 melanoma cells co-administered with BW001 and intraperitoneally injected into mice formed fewer tumor colonies in peritoneal cavities. This effect was related to the polyG motif and the full phosphorothioate modification backbone and enhanced by the existence of the CpG motif. Additional in vivo data showed that survival of tumor-bearing mice in the BW001 group was significantly prolonged, subcutaneous melanoma developed much more slowly, and lung dissemination colonies formed much less often than in mice inoculated with B16 melanoma cells only. The effect was CpG motif-dependent. These results suggest that BW001 may exert an integrated antitumor effect.

Studies of the carbohydrate-carbohydrate interaction between lactose and GM(3) using Langmuir monolayers and glycolipid micelles.

This paper describes studies of the carbohydrate-carbohydrate interaction (CCI) between micelles of a lactosyl lipid and monolayers of the glycosphingolipid GM(3). The lactose Lac.GM(3) interaction is involved in B16 melanoma cell adhesion and signaling processes, and a thorough understanding of the molecular details of this CCI is important for the design of new anti-adhesive and anti-metastatic agents. In this paper, we examine the influence of variations in divalent cations and subphase ionic strength on the Lac.GM(3) interaction. Our results indicate that, in the absence of divalent cations, the Lac.GM(3) CCI is strengthened at higher sodium chloride concentrations in the subphase. In contrast, when divalent cations are present in solution, the CCI is not as sensitive to ionic strength. These results suggest a role for both cation dependent as well as independent interactions in the Lac.GM(3) CCI.

Expression of the NG2 proteoglycan enhances the growth and metastatic properties of melanoma cells

The human homologue of NG2, the human melanoma proteoglycan (HMP), is expressed on most human melanomas. To investigate the role of this proteoglycan in melanoma progression, we have attempted to identify functionally important molecular ligands for NG2. Immunohistochemical analysis of cell lines that endogenously express NG2/HMP suggests that NG2/HMP associates with CD44 and α4β1 integrin, two molecules previously implicated in melanoma progression. Transfection of rat NG2 into the NG2-negative B16 mouse melanoma cell line also resulted in a highly colocalized pattern of expression between the transfected rat NG2 and the endogenously expressed mouse CD44 and α4β1 integrin molecules. In functional assays, expression of NG2 decreased the adhesion of B16 melanoma cells to CD44 monoclonal antibodies, hyaluronic acid, the C-terminal 40-kDa fibronectin fragment, and the CS1 fibronectin peptide, suggesting that NG2 may negatively modulate CD44- and α4β1-mediated binding events. Expression of NG2 increased the proliferation of melanoma cells in culture and increased tumorigenicity in vivo. Moreover, NG2 expression led to increased lung metastasis of B16F1 and B16F10 melanoma cells in experimental metastasis studies. Together, these studies demonstrate that NG2 is capable of modulating the adhesion, proliferation, and metastatic potential of melanoma cells. J. Cell. Physiol. 177:299–312, 1998. © 1998 Wiley-Liss, Inc.

Mannose receptor‐mediated endothelial cell activation contributes to B16 melanoma cell adhesion and metastasis in liver

The role of mannose receptors from hepatic sinusoidal endothelium (HSE) in liver colonization by B16 melanoma (B16M) cells was studied. The expression of high mannose-type oligosaccharides on the surface of B16M cells was enhanced by in vitro treatment with 1-deoximannojirimycin (1-DMM). There was a significant (P < 0.01) enhancement of hepatic metastasis when B16M cells were 1-DMM-treated before being intrasplenically injected into C57BL/6J mice. Intraperitoneal administration of 5 mg/kg recombinant human interleukin-1 receptor antagonist (rHuIL-1Ra) inhibited the 1-DMM-induced enhancement of metastasis. Expression of high mannose-type oligosaccharides on the surface of 1-DMM-treated B16M cells and their in vitro adhesion to the HSE was significantly correlated (R = 0.82). The addition of either 100 μg/ml mannan or paraformaldehyde (PFA)-fixed 1-DMM-treated B16M cells to cultured HSE for a period of 12 h significantly (P < 0.01) increased the release of IL-1β from the HSE compared to that liberated by the HSE incubated with either basal medium or PFA-fixed untreated B16M cells. The same HSE treatments also significantly (P < 0.01) increased the degree of adhesion of other B16M cells to HSE, being abrogated by anti-mouse vascular cell adhesion molecule-1 (VCAM-1) antibodies. The conditioned media from HSE cultures, activated by PFA-fixed, 1-DMM-treated B16M cells significantly (P < 0.01) increased B16M cell proliferation when compared to conditioned media from HSE cultures incubated with PFA-fixed, untreated B16M cells. Thus, 1-DMM treatment of B16M cells enhanced the development of hepatic metastasis by IL-1-dependent mechanisms. The mechanism is consistent with in vitro mannose receptor-mediated melanoma cell attachment to the HSE, which subsequently upregulates IL-1β release, VCAM-1-dependent adherence, and melanoma growth factor(s) release by HSE. J. Cell. Physiol. 174:322–330, 1998. © 1998 Wiley-Liss, Inc.

Sinusoidal endothelium release of hydrogen peroxide enhances very late antigen-4-mediated melanoma cell adherence and tumor cytotoxicity during interleukin-1 promotion of hepatic melanoma metastasis in mice.

The hepatic sinusoidal endothelium (HSE) releases large amounts of reactive oxygen species (ROS) in response to endotoxins and interleukin-1 (IL-1). Such pro-inflammatory mediators have been shown to promote hepatic metastasis. We have investigated the involvement of ROS released by IL-1-stimulated HSE in this promoting effect. Recombinant human interleukin-1 beta (rHuIL-1 beta) (5 micrograms/kg) was intravenously injected into C57BL/6J mice, and the hepatic metastasizing ability of B16 melanoma cells following intrasplenic injection was studied in the presence of ROS scavengers. rHuIL-1 beta-promoted hepatic metastases were significantly (P < .01) reduced by catalase (1 mg/kg) and enhanced by recombinant human superoxide dismutase (rHuSOD) (5 mg/kg). rHuIL-1 beta-stimulated HSE-conditioned medium (HSE-CM) significantly (P < .01) enhanced B16 melanoma cell adhesion to HSE compared with unstimulated HSE-CM, which in turn also significantly (P < .01) increased with melanoma cell adherence compared with basal medium. The addition of catalase completely abrogated proadhesive effects induced by rHuIL-1 beta-stimulated HSE-CM with respect to unstimulated HSE-CM, but did not affect the proadhesive effects induced by unstimulated HSE-CM over basal medium. The rat monoclonal antibody to mouse vascular cell adhesion molecule-1 (VCAM-1) significantly (P < .01) inhibited the enhanced melanoma cell adherence effects of both unstimulated and rHuIL-1 beta-stimulated HSE-CM, indicating that adherence was very late antigen-4 (VLA-4)-mediated. Not surprisingly, the percentage of VLA-4 expressing B16 melanoma cells significantly (P < .05) increased in response to unstimulated (21% of controls) and rHuIL-1 beta-stimulated (32% of controls) HSE-CM. Catalase addition abrogated these effects of rHuIL-1 beta-stimulated-HSE-CM. Melanoma cell damage was observed from the second hour of adhesion to HSE and significantly (P < .01) increased when the cells adhered to rHuIL-1 beta-stimulated HSE. This increase was abrogated by catalase. Cytolysis of the HSE was not observed during melanoma cell adhesion. Neither was the enhancement of B16 melanoma hydrogen peroxide production observed in response to rHuIL-1 beta. Thus, the effects of IL-1 in the liver may consist of a balance between the prometastatic effect of enhanced adherence to the HSE and the antimetastatic effect of H2O2-mediated cytotoxicity. Our results suggest that the enhancement of H2O2 production by the rHuIL-1 beta-stimulated HSE may contribute to the hepatic metastasis progression of ROS-resistant melanoma cells. Results in vitro indicate that this progression is associated with a H2O2-mediated increase in melanoma cell adhesion to HSE.

Interleukin-1-mediated H2O2 production by hepatic sinusoidal endothelium in response to B16 melanoma cell adhesion

We have examined H2O2 production by in vitro enriched hepatic sinusoidal endothelium (HSE) during interleukin-1 beta (IL-1 beta) stimulation and B16 melanoma cell adhesion. Production of H2O2 was quantified by flow cytometry and multiwell plate-scanning fluorimetry of intracellular 2', 7'-dichlorofluorescein (DCFH) oxidation in HSE. Under IL-1 beta treatment there was a 6-fold increase in endothelial cells producing H2O2 (67%) and a 4-fold augmentation in the Kupffer cell population (86%). The average H2O2 content per cell size unit significantly (P < 0.01) increased in endothelial cells (2.6-fold) and Kupffer cells (1.7-fold). In contrast to the homogeneity of Kupffer cells, H2O2 production intensity was largely heterogeneous in IL-1 beta-activated HSE. Enhancement of H2O2 production by IL-beta-treated HSE started at the 4th h and peaked 2-3 h later. The addition of increasing concentration of IL-1 beta to HSE for 4 h caused the progressive activation of H2O2 production by treated cells. The addition of 80 M excess of IL-1 receptor antagonist (IL-1 Ra) 10 min before IL-1 beta treatment abrogated IL-1 beta-mediated enhancement of H2O2. From the 2nd h of B16 melanoma adhesion to HSE there was significantly (P < 0.05) enhancement of H2O2 content in HSE. This activation increased 2.25-fold by the 3rd h of coculture and had reduced again by the 5th h. IL-1 Ra (80 ng/ml) given to HSE 10 min before melanoma cells abrogated the HSE response to melanoma cells. The addition of 1% paraformaldehyde (PFA)-fixed B16 melanoma cells to HSE did not affect H2O2 production response, indicating that HSE-activating agents were on the melanoma cell surface. Preincubation of B16 melanoma cells in the presence of 5 micrograms/ml anti-mouse IL-1 beta neutralizing antibody reduced the melanoma cell-induced HSE production of H2O2 by 80%. On the contrary, B16 melanoma cell-conditioned medium did not vary HSE production of H2O2 compared to control HSE. Western blot analysis of cytosolic and membrane sediments from B16 melanoma cells confirmed the presence of IL-1 beta (17.4 kDa) in both cell compartments. Thus, HSE responded to melanoma cell contact with a rapid production of H2O2. HSE activation was IL-1-dependent. This cytokine was directly provided to HSE by the cell surface of adhered melanoma cells.

Interleukin‐1‐mediated H2O2 production by hepatic sinusoidal endothelium in response to B16 melanoma cell adhesion

Interleukin‐1‐mediated H2O2 production by hepatic sinusoidal endothelium in response to B16 melanoma cell adhesion

Inhibition of Radiation-Enhanced Expression of Integrin and Metastatic Potential in B16 Melanoma Cells by a Lipoxygenase Inhibitor

Low-dose gamma radiation stimulates expression of phenotypic characteristics in B16 melanoma cells which regulate metastatic potential. A transient increase in the expression of an integrin receptor (alpha IIb beta 3) was observed after exposure of B16 melanoma cells to 0.25 to 2.0 Gy of gamma radiation. This increased receptor expression resulted in enhanced adhesion of tumor cells to fibronectin in vitro and increased experimentally induced metastasis in vivo. In this report, we determined a role for the 12-lipoxygenase metabolite, 12-HETE, in radiation-enhanced metastasis. A significant increase in biosynthesis of 12-HETE in B16 melanoma cells was detected < 5 min after exposure to 0.5 Gy gamma radiation. We then determined that radiation-enhanced expression of alpha IIb beta 3 integrin and adhesion of B16 melanoma cells to fibronectin in vitro and metastasis in vivo were reduced by treatment of the cells with the lipoxygenase inhibitor NDGA prior to irradiation. These findings suggest that low-dose radiation, at levels comparable to those used in fractionated or hyper-fractionated radiotherapy, increases the metastatic potential of surviving tumor cells via a rapid and transient alteration in lipoxygenase metabolism of arachidonic acid and surface expression of an integrin receptor.

Cytostatin, a novel inhibitor of cell adhesion to components of extracellular matrix produced by Streptomyces sp. MJ654-NF4. I. Taxonomy, fermentation, isolation and biological activities.

Cytostatin has been identified as a novel inhibitor of cell adhesion to components of extracellular matrix (ECM) in cultured broth of Streptomyces sp. MJ654-NF4. Though cytostatin did not inhibit EL-4 cell adhesion to ECM components such as laminin and fibronectin; it inhibited the adhesion of B16 melanoma cells to laminin and collagen type IV but not to fibronectin. It exhibited antimetastatic activity on B16 melanoma cells in mice. The cytotoxicity of cytostatin are also reported.

Cell adhesion in a dynamic flow system as compared to static system. Glycosphingolipid-glycosphingolipid interaction in the dynamic system predominates over lectin- or integrin-based mechanisms in adhesion of B16 melanoma cells to non-activated endothelial cells.

Initial adhesion of B16 melanoma variants to non-activated endothelial cells is mediated through specific interaction between GM3 (NeuAc alpha 2----3Gal beta 1----4Glc beta 1----Cer) expressed on melanoma cells and lactosylceramide (LacCer, Gal beta 1----4Glc beta 1----Cer) expressed on endothelial cells. This adhesion is predominant over integrin- or lectin-mediated adhesion in a dynamic flow experimental system employing a parallel plate laminar flow chamber (Lawrence, M. B., Smith, C. W., Eskin, S. G., and McIntire, L. V. (1990) Blood 75, 227-237). In this system, a tumor cell suspension flows over a glass plate coated with glycosphingolipid, lectin, or fibronectin, and adhesion is recorded on videotape. These conditions were designed to mimic the microvascular environment in which tumor metastatic deposition takes place. In contrast, lectin- and fibronectin-based mechanisms are predominant in previously used static adhesion systems. Under static conditions, the relative degree of adhesion of the four B16 variants to endothelial cells or to LacCer-coated plates was the same as their relative degree of GM3 expression (i.e. BL6 approximately F10 greater than F1 greater than WA4), and adhesion was inhibited in the presence of methyl-beta-lactoside, or liposomes containing LacCer or GM3. Adhesion was also inhibited by pretreatment of B16 cells with anti-GM3 antibody DH2 or sialidase and by pretreatment of endothelial cells with anti-LacCer antibody T5A7. Under dynamic flow conditions, WA4 cells did not adhere to mouse endothelial cells at high shear stress (greater than 2.5 dynes/cm2) but did adhere at lower shear stress. In contrast, BL6 and F10 cells adhered strongly at both low and high shear stress. BL6 cell adhesion to endothelial cells at both low and high shear stress was inhibited in the presence of antibody DH2, ethyl-beta-lactoside, or lactose, as well as by pretreatment of BL6 cells with sialidase. Thus, some clear differences, as well as similarities, in cell adhesion under static versus dynamic conditions are demonstrated. These findings suggest that melanoma cell adhesion to endothelial cells, based on GM3/LacCer interaction, initiates metastatic deposition, which may trigger a series of "cascade" reactions leading to activation of endothelial cells and expression of Ig family or selectin receptors, thereby promoting adhesion and migration of tumor cells.

Involvement of endothelial cell mannose-binding surface molecules in the adhesion of B16 melanoma cells to mouse liver sinusoids : F. Vidal-Vanaclocha, A. Asumendi, M.A. Rocha, E. Barberá-Guillem. Dpt. Cell Biol. & Morphol. Sci, Sch. Med. & Dent., Univ. Basque Country, Leioa, 48940-Vizcaya, Spain

Involvement of endothelial cell mannose-binding surface molecules in the adhesion of B16 melanoma cells to mouse liver sinusoids : F. Vidal-Vanaclocha, A. Asumendi, M.A. Rocha, E. Barberá-Guillem. Dpt. Cell Biol. & Morphol. Sci, Sch. Med. & Dent., Univ. Basque Country, Leioa, 48940-Vizcaya, Spain

Modulation of melanoma cell adhesion to basement membrane components by retinoic acid.

The effect of retinoic acid pretreatment on metastatic B16 melanoma cell adhesion in serum-free medium to tissue culture plastic precoated with fibronectin, laminin/nidogen, type I and type IV collagen was examined. Both control cells grown to subconfluence and cells treated with 10(-6) M-retinoic acid adhered and spread rapidly on fibronectin (greater than 75% following 1 h of incubation) but adhered poorly to type I collagen (less than 15%). Control cells adhered to laminin/nidogen (greater than 35%), type IV collagen (greater than 58%) and type IV collagen plus laminin/nidogen (greater than 80%), while retinoic acid-treated cells showed a reduced ability to attach and spread on these substrata, the number of adherent cells being reduced by 61% on laminin/nidogen, by 19% on type IV collagen, and by 41% on type IV collagen plus laminin/nidogen following 1 h of incubation. The minimum concentration of retinoic acid required to yield an effective reduction in adhesion was 10(-7) M for type IV collagen and 10(-10) M for laminin/nidogen. Melanoma cells harvested at low density showed a reduced adhesion to laminin/nidogen and type IV collagen compared to that of subconfluent control cultures, but also showed a reduced adhesion to fibronectin. The effect of retinoic acid on cell adhesion was not, however, due to reduced cell density, as the cells were seeded so that control and retinoic acid-treated cultures were of a similar density when harvested.

The role of fibronectin in adhesion of metastatic melanoma cells to endothelial cells and their basal lamina

Vascular endothelial cells synthesize an extracellular matrix or basal lamina composed of collagens, proteoglycans and glycoproteins such as fibronectin (FN). Using affinity-purified anti-FN, we have examined the role of FN in adherence of metastatic B16 melanoma cells to endothelial cell monolayers which lack FN on apical cell surfaces and to their basal lamina which contains FN. B16 melanoma cells, which do not contain significant amounts of FN, attached at much higher rates to endothelial basal lamina and polyvinyl-immobilized FN compared with intact endothelial cell monolayers. Anti-FN failed to inhibit attachment of melanoma sublines of low (B16-F1) or high (B16-F10) metastatic potential to intact endothelial cell monolayers, inhibited slightly B16 cell attachment to basal lamina and completely abolished attachment of B16 cells to polyvinyl-immobilized FN. The antibiotic tunicamycin which inhibits glycosylation of B16 cell surface glycoproteins and blocks experimental metastasis [18] inhibited B16 attachment to endothelial cells, basal lamina and immobilized FN. The results suggest that FN mediates, only in part, the adhesion of B16 melanoma cells to basal lamina through glycoprotein receptors on B16 cells.