Adhesion Of B16-F10 Melanoma Cells Research Articles

GPR4 decreases B16F10 melanoma cell spreading and regulates focal adhesion dynamics through the G13/Rho signaling pathway

The effect of acidosis, a biochemical hallmark of the tumor microenvironment, on cancer progression and metastasis is complex. Both pro- and anti-tumorigenic effects of acidosis have been reported and the acidic microenvironment has been exploited for specific delivery of drugs, imaging agents, and genetic constructs into tumors. In this study we investigate the spreading and focal adhesion of B16F10 melanoma cells that are genetically engineered to overexpress the pH-sensing G protein-coupled receptor GPR4. By using cell attachment assays we found that GPR4 overexpression delayed cell spreading and altered the spatial localization of dynamic focal adhesion complex, such as the localization of phosphorylated focal adhesion kinase (FAK) and paxillin, at acidic pH. The potential G-protein and downstream signaling pathways that are responsible for these effects were also investigated. By using the Rho inhibitor CT04 (C3 transferase), the Rho-associated kinase (ROCK) inhibitors Y27632 and thiazovivin, the myosin light chain kinase (MLCK) inhibitor staurosporine or a G12/13 inhibitory construct, cell spreading was restored whereas the inhibition and activation of the Gq and Gs pathways had little or no effect. Altogether our results indicate that through the G12/13/Rho signaling pathway GPR4 modulates focal adhesion dynamics and reduces cell spreading and membrane ruffling.

Marine algal fucoxanthin inhibits the metastatic potential of cancer cells

Metastasis is major cause of malignant cancer-associated mortality. Fucoxanthin has effect on various pharmacological activities including anti-cancer activity. However, the inhibitory effect of fucoxanthin on cancer metastasis remains unclear. Here, we show that fucoxanthin isolated from brown alga Saccharina japonica has anti-metastatic activity. To check anti-metastatic properties of fucoxanthin, in vitro models including assays for invasion, migration, actin fiber organization and cancer cell–endothelial cell interaction were used. Fucoxanthin inhibited the expression and secretion of MMP-9 which plays a critical role in tumor invasion and migration, and also suppressed invasion of highly metastatic B16-F10 melanoma cells as evidenced by transwell invasion assay. In addition, fucoxanthin diminished the expressions of the cell surface glycoprotein CD44 and CXC chemokine receptor-4 (CXCR4) which play roles in migration, invasion and cancer–endothelial cell adhesion. Fucoxanthin markedly suppressed cell migration in wound healing assay and inhibited actin fiber formation. The adhesion of B16-F10 melanoma cells to the endothelial cells was significantly inhibited by fucoxanthin. Moreover, in experimental lung metastasis in vivo assay, fucoxanthin resulted in significant reduction of tumor nodules. Taken together, we demonstrate, for the first time, that fucoxanthin suppresses metastasis of highly metastatic B16-F10 melanoma cells in vitro and in vivo.

Anti-metastatic Effects on B16F10 Melanoma Cells of Extracts and Two Prenylated Xanthones Isolated from Maclura amboinensis Bl. Roots

Inhibitory effects of Maclura amboinenesis Bl, one plant used traditionally for the treatment of cancers, on metastatic potential of highly metastatic B16F10 melanoma cells were investigated in vitro. Cell proliferation was assessed using the MTT colorimetric assay. Details of metastatic capabilities including invasion, migration and adhesion of B16F10 melanoma cells were examined by Boyden Chamber invasion and migration, scratch motility and cell attachment assays, respectively. The results demonstrated that n-hexane and chloroform extracts exhibited potent anti-proliferative effects (p<0.01), whereas the methanol and aqueous extracts had less pronounced effects after 24 h exposure. Bioactivity-guided chromatographic fractionation of both active n-hexane and chloroform extracts led to the isolation of two main prenylated xanthones and characterization as macluraxanthone and gerontoxanthone-I, respectively, their structures being identified by comparison with the spectral data. Interestingly, both exhibited potent effective effects. At non-toxic effective doses, n-hexane and chloroform extracts (10 and 30 μg/ml) as well as macluraxanthone and gerontoxanthone-I (3 and 10 μM) significantly inhibited B16F10 cell invasion, to a greater extent than 10 μM doxorubicin, while reducing migration of cancer cells without cellular cytotoxicity. Moreover, exposure of B16F10 melanoma cells to high concentrations of chloroform (30 μg/ml) and geratoxanthone-I (20 μM) for 24 h resulted in delayed adhesion and retarded colonization. As insights into mechanisms of action, typical morphological changes of apoptotic cells e.g. membrane blebbing, chromatin condensation, nuclear fragmentation, apoptotic bodies and loss of adhesion as well as cell cycle arrest in the G1 phase with increase of sub-G1 cell proportions, detected by Hoechst 33342 staining and flow cytometry were observed, suggesting DNA damage and subsequent apoptotic cell death. Taken together, our findings indicate for the first time that active n-hexane and chloroform extracts as well as macluraxanthone and gerontoxanthone-I isolated from Maclura amboinensis Bl. roots affect multistep of cancer metastasis processes including proliferation, adhesion, invasion and migration, possibly through induction of apoptosis of highly metastatic B16F10 melanoma cells. Based on these data, M. amboinensis Bl. represents a potential candidate novel chemopreventive and/or chemotherapeutic agent. Additionally, they also support its ethno-medicinal usage for cancer prevention and/or chemotherapy.

Pentoxifylline modulates cell surface integrin expression and integrin mediated adhesion of B16F10 cells to extracellular matrix components

Our previous studies demonstrated that Pentoxifylline (PTX), a phosphodiesterase inhibitor could inhibit the lung homing of B16-F10 melanoma cells in C57BL/6 mice. In this study we have looked at the effect of PTX on cell surface integrin expression and integrin mediated adhesion of B16-F10 melanoma cells. B16F10 cells treated with PTX when injected through the tail vein of mice showed a 75% reduction in pulmonary nodules as compared to control untreated cells. PTX brought about a significant reduction in the integrin mediated adhesion of F10 cells to Fibronectin and Vitronectin (58.75% + 3.4 S.E and 60% + 1.7 S.E respectively if control was considered as 100 %). This inhibition in adhesion was evident upto 4 hours only and treatment for 24 hours brought about an increase in adhesion (135.5 % + 0.5 S.E). Flow cytometric analysis showed higher surface expressions of αv, α5 and αIIb integrin subunits in B16-F10 as compared to the low metastatic cell line B16-F1 suggesting a role for these integrins in determining the metastatic potential. PTX brought about a significant decrease in the cell surface expression of α5, αIIb and β1integrin subunits but not that of the αv subunit on B16-F10 cells. PTX also brought about a reduction in the total cellular protein levels of β1 and αv integrin subunits. Various isoforms of Protein Kinase C (PKC) has been shown to regulate integrin expression, localization and activity. Hence we looked at the effect of PTX on total cellular PKC activity. PTX brought about a significant reduction in total cellular PKC activity (82.66 + 0.593). Collectively our results indicate that the antimetastatic action of PTX is mediated, at least in part through its effects on adhesion and the surface expression of specific integrin receptors.

Effect of a hyaluronan synthase suppressor, 4-methylumbelliferone, on B16F-10 melanoma cell adhesion and locomotion

Hyaluronan (HA) is a ubiquitous, major component of the extracellular matrix. It is involved in cell adhesion and locomotion, and hence in tumor metastasis. We have previously reported that 4-methylumbelliferone (MU) inhibits HA synthesis and may be a useful tool for examining the functions of HA. We here demonstrate that the formation of cell surface HA by melanoma cells and its release into the culture medium are inhibited by MU. Adhesion and locomotion assays revealed that the adhesion and locomotion of melanoma cells were dose-dependently inhibited by MU. Conversely, treatment with exogenous HA enhanced both adhesion and locomotion. Thus, preventing the formation of cell surface HA reduced both the adhesion and locomotion of melanoma cells, suggesting that MU may act as an inhibitor of tumor metastasis.

Vascular Cell Adhesion Molecule-1 Is a Key Adhesion Molecule in Melanoma Cell Adhesion to the Leptomeninges

Leptomeningeal metastases occur in up to 8% of patients with systemic malignancies and have a poor prognosis. A better understanding of the pathophysiologic processes underlying leptomeningeal metastases is needed for more effective treatment strategies. We hypothesized that tumor cells will have to adhere to the well-vascularized leptomeninges, because the cerebrospinal fluid lacks nutrients and growth factors for efficient tumor cell proliferation. Specific receptor-ligand interactions, which are unknown until now, will mediate this adhesion process. We determined the growth characteristics of B16F-10 melanoma cells in cerebrospinal fluid. The expression levels of specific adhesion molecules on both mouse leptomeningeal cells (MLMC) and murine B16F-10 melanoma cells were measured by immunofluorescence flow cytometry. We used mAbs to determine the function of these specific adhesion molecules on B16F-10 melanoma cell adhesion to a leptomeningeal cell layer under static and (cerebrospinal fluid-like) flow conditions. B16F-10 melanoma cells did not proliferate in cerebrospinal fluid because of a lack of nutrients and growth factors. MLMC expressed low levels of vascular cell adhesion molecule-1 (VCAM-1), intercellular adhesion molecule-1 (ICAM-1), β1- and β3-integrin subunits, and CD44. VCAM-1 expression on MLMC was shown to be up-regulated by TNF-α. Blocking VCAM-1 on the MLMC with a mAb resulted in a 60% inhibition of melanoma cell adhesion to a leptomeningeal cell layer under flow but not under static conditions. No additive inhibitory effect on melanoma cell adhesion was found by concomitant blocking of the β1- and β3-integrin subunits and CD44 with mAbs. Our experiments indicate that cerebrospinal fluid does not support B16F-10 melanoma cell proliferation, suggesting the need for melanoma cell adhesion to the well-vascularized leptomeninges. VCAM-1, expressed on MLMC, is an important mediator of in vitro melanoma cell adhesion under (cerebrospinal fluid-like) flow conditions.

Antimetastatic and antitumor effects of benzoquinonoid AC7-1 from Ardisia crispa.

An antimetastatic and cytostatic substance, termed AC7-1, was isolated from Ardisia crispa and identified as a benzoquinonoid compound, 2-methoxy-6-tridecyl-1,4-benzoquinone. It was originally characterized as the potent PAF (platelet-activating factor) receptor-binding antagonist with nonspecific antiplatelet effects on platelet aggregation induced by various agonists including PAF, ADP, thrombin and collagen. The nonspecific antiaggregatory properties of AC7-1 drew our interest given its possible relationship in integrin receptor-binding antagonistic activity. The integrin receptor plays an important role in metastasis and thrombosis as the cell surface transmembrane protein. Based on the aforementioned facts, the antimetastatic activities of AC7-1 were examined using various in vitro and in vivo metastasis assays. AC7-1 strongly blocked B16-F10 melanoma cell adhesion to extracellular matrix (ECM) and B16-F10 melanoma cell invasion. AC7-1 also remarkably inhibited pulmonary metastasis and tumor growth in vivo. AC7-1 inhibited B16-F10 melanoma cell adhesion to only specific synthetic peptides including RGDS. These findings suggest that antimetastatic activities of AC7-1 can be caused by blocking integrin-mediated adherence. We found AC7-1 to be a potential candidate for the development of a new antimetastatic drug.

Suppressive Mechanism of Salmosin, a Novel Disintegrin in B16 Melanoma Cell Metastasis

We have previously reported that salmosin, a novel disintegrin, was isolated from Korean snake (Agkistrodon halys brevicaudus) venom and significantly inhibited solid tumor growth in mice by perturbation of tumor-specific angiogenesis via blocking αvβ3 integrin expressed on vascular endothelial cells. In this study, we investigated the functional specificity of salmosin in tumor cell metastasis. Recombinant salmosin expressed in E. coli that has the RGD sequence markedly inhibited both B16F10 melanoma cell adhesion to the extracellular matrix proteins as well as B16F10 melanoma cell invasion through Matrigel-coated filter. The inhibition by salmosin can be caused by blocking integrins expressed on the surface of B16F10 melanoma cells. Salmosin significantly inhibited the proliferation of B16F10 melanoma cells on the plate coated with collagen I in a dose-dependent manner. In vivo B16F10 melanoma experimental metastasis, salmosin showed remarkable significant inhibitory effect on lung tumor colonization in a concentration-dependent manner. These results clearly demonstrate that antimetastatic activity of salmosin resulted from blocking the integrin-mediated adherence and αvβ3 integrin-mediated proliferation of the melanoma cells.

Synthesis and activity of partial retro-inverso analogs of the antimetastatic laminin-derived peptide, YIGSR-NH2.

This paper describes the synthesis and biological evaluation of six partial retro-inverso peptidomimetic analogs of YIGSR-NH2, a synthetic peptide from the beta 1 chain of laminin, which has antimetastatic activity. The intent was to improve the antimetastatic potency of YIGSR-NH2 by limiting the in vivo enzymatic degradation through the incorporation of fraudulent peptide bonds. We have prepared the following retro-inverso peptides, Tyr-Ile-Gly-Ser-gArg-CHO (1), Tyr-gIle-mGly-Ser-Arg-NH2 (2), Tyr-gIle-mGly-Ser-gArg-CHO (3), gTyr-D-rIle-mGly-Ser-Arg-NH2 (4), Tyr-Ile-Gly-gSer-D-rArg-CHO (5) and Tyr-gIle-rGly-D-rSer-D-rArg-CHO (6). In vitro assays for B16F10 melanoma cell adhesion showed no significant activity for these six peptides. Peptides 1-3, 5 and 6 were further tested, in vivo, for their ability to inhibit tumor metastases to the lung in mice injected in the tail vein with B16F10 melanoma cells. All five of the retro-inverso peptides tested showed statistically significant inhibition of metastasis, but the most active peptides were 5 and 6, which showed 57 and 69% inhibition of metastasis, respectively.

Lu-ECAM-1-mediated adhesion of melanoma cells to endothelium under conditions of flow.

Lu-ECAM-1 is a lung-derived, venular endothelial cell adhesion molecule. It promotes the selective adhesion of lung-metastatic B16-F10 melanoma cells to endothelium under static conditions and mediates colonization of the lungs by the same tumor cells. To test whether Lu-ECAM-1 by itself is sufficient to cause vascular arrest of B16-F10 cells, we measured here under conditions of flow tumor cell adhesion to endothelia that express different amounts of Lu-ECAM-1 on their surfaces. At physiological shear stresses, adhesion of B16-F10 melanoma cells to endothelia correlates positively with the amount of Lu-ECAM-1 expression on the endothelial cell surface and inversely with the level of the applied shear stress. Tumor cell trajectories are biphasic; i.e., B16-F10 melanoma cells initially move along the endothelial surface with a velocity similar to the theoretical velocity, then arrest within a fraction of a second. Arrest is permanent for most B16-F10 melanoma cells at all shear stresses tested. Tumor cells never engaged in a rolling motion prior to arrest. Masking of the Lu-ECAM-1 ligand on the surface of B16-F10 melanoma cells with soluble Lu-ECAM-1 impedes arrest of tumor cells on the surface of the test endothelium. Purified Lu-ECAM-1 also mediates B16-F10 arrest, but arrest is mostly transient at shear stresses of 0.59 dynes/cm2 and higher, implying adhesion by single receptor/ligand bonds. Our data suggest that Lu-ECAM-1 plays a critical role in the recognition and initial arrest of murine melanoma cells in lung venules.

Inhibition of metastatic cell colonization in murine lungs and tumor-induced morbidity by non-peptidic Arg-Gly-Asp mimetics.

The spreading and colonization of tumor cells require their migration to metastatic sites via blood vessels. To penetrate blood-vessel walls, cells, including malignant ones, must recognize and associate with the sub-endothelium extracellular matrix (ECM) and its glycoproteins. Recognition of ECM-glycoproteins, such as fibronectin (FN) and vitronectin (VN), is mediated by integrin receptors expressed on various cell types, including platelets, leukocytes and tumor cells. The Arg-Gly-Asp (RGD)-containing peptide, a major adhesive ligand of ECM, is present in various plasma and matrix glycoproteins, such as FN and VN. Non-peptidic mimetics of RGD, consisting of carboxylate and guanidinium groups of Asp and Arg divided by a linear atom spacer, express a high affinity for the alpha IIb-beta 3 integrin and inhibit platelet aggregation. Herein, the ability of RGD mimetics to inhibit adhesive interactions between tumor cells and RGD, and tumor progression in vivo, was examined. RGD-containing peptides and the RGD mimetic, compound SF-6,5, but not the Arg-Gly-Glu (RGE) peptide or the corresponding mimetic, specifically inhibited B16-F10 melanoma cell adhesion to immobilized VN and FN. Daily administration in vivo of SF-6,5 to mice inhibited the formation of B16-F10 colonies in experimental and spontaneous models of metastases. Moreover, SF-6,5 could prevent mouse death caused by massive colonization of tumor cells in the lungs. The therapeutic effect of RGD-containing peptides on tumor metastasis formation was marginal, probably due to the small amounts used, and its susceptibility to proteolysis in situ. Thus, non-peptidic mimetics of small adhesive epitopes may provide a novel therapeutic tool to prevent an adverse pathological event involving integrin-dependent cell-cell and cell-ECM interactions.

Identification of two distinct regions of the type III connecting segment of human plasma fibronectin that promote cell type-specific adhesion.

The principal region of the human plasma fibronectin molecule mediating the adhesion of melanoma cells appears to be the alternatively spliced type III connecting segment (IIICS (Humphries, M. J., Akiyama, S. K., Komoriya, A., Olden, K., and Yamada, K. M. (1986a) J. Cell Biol., in press]. A series of overlapping synthetic peptides spanning the entire IIICS (CS peptides) were examined for their effects on B16-F10 melanoma cell adhesion to the parent fibronectin molecule. Two nonadjacent CS peptides, designated CS1 and CS5, were inhibitory. In contrast, neither inhibited fibronectin-mediated spreading of fibroblastic baby hamster kidney cells. When N-terminal cysteine derivatives of the CS peptides were conjugated to IgG by covalent cross-linking with N-succinimidyl-3(2-pyridyldithio)propionate, both the CS1 and CS5 conjugates promoted B16-F10 melanoma cell spreading. All conjugates were inactive for spreading of baby hamster kidney cells, confirming the cell type specificity of the IIICS adhesion site. Determination of the amounts of CS peptide required to support melanoma cell adhesion revealed that the activity of CS1 was only 2.4-fold lower than that of the intact fibronectin molecule. CS5 was approximately 320-fold less active than fibronectin, suggesting that the CS1 region may be the major site of interaction with the melanoma cell surface. The adhesion-promoting activities of CS1-IgG and CS5-IgG were additive as were the inhibitory activities of the free peptides for B16-F10 cell spreading on fibronectin. These findings suggest that both regions of the IIICS can function separately or together in mediating the interaction of melanoma cells with fibronectin. Since CS1 and CS5 are each found in separate alternatively spliced regions of the IIICS, it is conceivable that the adhesion-promoting activity of fibronectin for different cell types may be under complex regulation.