Inhibition of invasion and experimental metastasis of murine melanoma cells by human soluble thrombomodulin

Thrombomodulin (TM) is an anticoagulant molecule expressed on the endothelial cell surface and soluble TM antigen, which is present in human plasma and urine, represents the products of limited proteolytic cleavage of cell-surface TM. Recently, it was demonstrated that TM is also expressed on the surface of several tumor cells and the expression level of TM negatively correlated with malignancy in cancer. We investigated the effect of soluble TM isolated from human urine (uTM) on the invasion and metastasis of murine melanoma cells (B16F10 cells) through a reconstituted basement membrane (Matrigel) and in a murine model of experimental lung metastasis. Matrigel reconstituted with uTM inhibited the invasion of B16F10 cells in a dose-dependent manner in a range from 10 to 1000 ng/ml uTM as compared with the control Matrigel without uTM. The inhibitory action of uTM was not altered in the presence of an excess amount of hirudin, an inhibitor of thrombin proteolytic activity, but abolished in the presence of anti-human TM IgG. Matrigel reconstituted with thrombin (1 NIH unit/ml) enhanced the invasion level of cells by 1.5-fold relative to the control Matrigel without thrombin. The thrombin-enhanced invasion of B16F10 cells was repressed by addition of hirudin (10 units/ml) or uTM (100 ng/ml) into the Matrigel. Matrigel reconstituted with hirudin (10 units/ml) and uTM (100 ng/ml) additionally accelerated the inhibitory activity of hirudin or uTM on the thrombin-enhanced invasion of B16F10 cells. Moreover, metastatic colonies formed in the lungs of mice injected intravenously with B16F10 cells were significantly reduced by injection of uTM once a day up to 2 days after co-injection of uTM with the cells. These results suggested that Matrigel reconstituted with uTM inhibited the invasion of B16F10 cells in vitro through a thrombin-independent mechanism and the injection of uTM suppressed experimental lung metastasis of the cells in mice.     

Inhibition of murine melanoma experimental metastasis by recombinant desulfatohirudin, a highly specific thrombin inhibitor

Recombinant desulfatohirudin (r-hirudin), a highly specific inhibitor of thrombin, was examined to determine whether it would inhibit production of experimental lung metastasis by B16-F10 melanoma cells. In in vitro assays using mouse plasma, the high level of procoagulant activity in B16-F10 cells was significantly inhibited by r-hirudin in a dose-dependent manner. From 15 to 120 min after s.c. administration into C57BL/6 mice, r-hirudin (10 mg/kg) markedly prolonged clotting time in a time course pattern that directly correlated with that of blood distribution of 125I-labeled r-hirudin. The production of experimental lung metastasis by B16-F10 cells was significantly inhibited by r-hirudin administered s.c. at time points ranging from 120 min before to 60 min after tumor cell inoculation with the most significant effects found in mice given r-hirudin 15 or 2 min before the i.v. injection of tumor cells. The organ distribution of [125I]IdUrd-labeled tumor cells demonstrated a clear difference in the lungs of mice treated with r-hirudin and the lungs of control mice, and these differences directly correlated with the number of lung tumor colonies found 3 weeks later. The inhibition of lung metastasis was not due to direct antitumor effects of r-hirudin. These results suggest that inhibition of coagulation events by r-hirudin significantly inhibit experimental lung metastasis during a critical time of 60 min after the entry of tumor cells into the circulation.     

Brain surface invasion and metastasis of murine malignant melanoma variants

Mouse B16 melanoma sublines were selected sequentially for their abilities to colonize brain meninges and leptomeninges of C5713L/6 mice. After 14 selections subline 1316-B14b was established that formed significantly more brain tumor colonies than the parental B16 line. Examination of brains at various times after intravenous or intra-arterial injection of B16 cells by electron microscopy revealed that B14b melanoma cells lodged in small brain blood vessels, proliferated and invaded through vessel walls into brain parenchyma and also along small blood vessels at perivascular sites. Invasion into brain parenchyma was characterized by extension of melanoma cell filopodia resulting in fragmentation and sometimes enfulgment of glial and neural cells.Analysis of cell surface proteins of B16 melanoma sublines revealed increased exposure of a M_r 90 000 glycoprotein on the high brain-colonizing cells. Antibodies against the M_r 90 000 glycoprotein reacted with a variety of human melanoma cell lines and with some fetal and adult tissues, indicating that this melanoma-associated component is not species-, tumor- or tissue-specific. The glycoprotein could be a cell surface receptor important in the survival and growth properties of melanoma cells in brain microenvironments.     

Inhibition of experimental metastasis and extracellular matrix degradation by butanol extracts from B16-F1 murine melanoma

We previously demonstrated that noncytolytic butanol extraction of B16 melanoma cells can increase the number of experimental lung metastases, and that brief incubation of the extracted cells with the extracted moieties reduces metastatic phenotype. This study examined the possibility that the extracted components are endogenous inhibitors of tumor cell surface-associated, degradative enzymes. The activity was found to be tumor associated, since only tumor extracts could reduce the number of experimental lung metastases of a variety of solid tumors. The activity in crude butanol extracts of B16-F1 that modulated the metastatic phenotype of extracted B16-F10 was partially purified by preparative isoelectric focusing and high-performance gel permeation chromatography. Incubation of extracted B16-F10 cells with low (Mr 2,000-10,000) molecular weight materials focusing in the pH 5.6 to 5.8 region of the preparative isoelectric focusing gradient significantly reduced the number of experimental lung foci. Ampholines alone had no effect. Evidence that the extracted moiety might be an endogenous enzyme inhibitor was obtained with the use of the subendothelial matrix degradation assay, wherein B16-F10 cells digest 35S-labeled heparan sulfate proteoglycan. The same materials that reduced the metastatic potential of butanol-extracted B16-F10 cells also inhibited extracellular matrix degradation by 30 to 85%, as well as the activity of partially purified heparanase (endo-beta-glucuronidase). The metalloproteinase inhibitor 1,10-phenanthroline and the heparanase inhibitor heparin partially (30 to 50%) blocked extracellular matrix degradation. Conversely, inhibitors of serine, thiol, acid, and other proteases had little or no effect on extracellular matrix degradation. These data provide evidence that an endogenous, heat-stable inhibitor of cell surface degradative enzymes such as heparanase may play a role in hematogenous metastasis, and support the hypothesis that butanol extraction activates some of these surface enzymes by removing the endogenous inhibitors.     

Inhibition by sulfated chitin derivatives of invasion through extracellular matrix and enzymatic degradation by metastatic melanoma cells

We have investigated the effects of sulfated chitin derivatives and heparin on the invasion of B16-BL6 melanoma cells through reconstituted basement membrane Matrigel which contains laminin, type IV collagen, heparin sulfate proteoglycan, and entactin. 6-O-sulfated chitin (S-chitin) and 6-O-sulfated and carboxymethyl chitin (SCM-chitin) significantly inhibited the penetration of tumor cells through Matrigel in parallel with the increased degree of sulfation. However, 6-O- and N-sulfated but partially N-deacetylated chitin derivative (SCM-chitosan) and CM-chitin had no effect. SCM-chitin with a high degree of sulfation (SCM-chitin III), which exhibited fairly low levels of anticoagulant activity, was more effective than intact heparin. SCM-chitin III and heparin were also shown to block the attachment and migration of tumor cells to laminin-coated substrates, which are considered to be involved in tumor invasion. The inhibition of cell attachment and migration by SCM-chitin III and heparin is likely to depend upon their specific binding to laminin molecules (possibly the heparin-binding domain). Degradation of heparan sulfate by heparanase was inhibited by SCM-chitin III and heparin in a dose-dependent manner. Surprisingly, SCM-chitin III could inhibit type IV collagenolytic activity of tumor cells more potently than heparin. Thus, nontoxic SCM-chitin III of low anticoagulant properties may provide a promising basis for the prevention of cancer metastasis.     

Inhibition of experimental metastasis of murine fibrosarcoma cells by oligopeptide analogues to the fibronectin cell-binding site

We have analyzed the effect of the synthetic oligopeptides Gly-Arg-Gly-Asp-Ser-Pro (GRGDSP) and Gly-Arg-Gly-Glu-Ser-Pro (GRGESP), analogues to the fibronectin cell-binding sequence, on the formation of experimental lung metastasis. SR-BALB were injected alone or in conjunction with GRGDSP or GRGESP in the tail vein of BALB/c mice. Co-injection with GRGESP reduced by approximately 70% the number of metastatic colonies per mouse. However, co-injection with the closely related peptide GRGDSP, containing the conservative substitution Glu/Asp, did not affect metastatic behavior. GRGESP peptide anti-metastatic activity was not due to a toxic effect on tumor cells or on mice. In vitro adhesion assays testing for a possible effect of the peptide on cell-matrix interactions indicated that the GRGESP peptide did not affect cell adhesion to the matrix proteins tested.     

Inhibition of tumorigenicity and metastasis of human melanoma cells by anti-cathepsin L single chain variable fragment

We demonstrated previously that the switch from nonmetastatic to highly metastatic phenotype of human melanoma cells is directly related to secretion of procathepsin L form. This cysteine proteinase was identified on the basis of its property to cleave human C3, the third component of complement. In an attempt to control procathepsin L secretion, we have recently generated an anti-cathepsin L single chain variable fragment (ScFv) from an anti-cathepsin L monoclonal antibody generated against recombinant cathepsin L. We herein selected clones stably transfected with this anti-cathepsin L ScFv and analyzed them for changes in tumor growth and metastasis. We show that in stably transfected clones, anti-cathepsin L ScFv strongly inhibited the secretion of procathepsin L without modifying the intracellular amount or processing pattern of cathepsin L forms. Confocal analysis demonstrated colocalization of endogenous cathepsin L and anti-cathepsin L ScFv. In addition, expression of this ScFv strongly inhibited generation of tumor and metastasis by these human melanoma clones in nude mice. In vivo, the anti-cathepsin L ScFv-transfected cells produced tumors with decreased vascularization (angiogenesis) concomitant with increased apoptosis of tumor cells. Matrigel assay also demonstrated that melanoma invasiveness was completely abolished. Thus, this is the first demonstration that anti-cathepsin L ScFv could be used to inhibit the tumorigenic and metastatic phenotype of human melanoma, depending on procathepsin L secretion, and could therefore be used as a molecular tool in a therapeutic cellular approach.     

Inhibition of cytokine-induced microvascular arrest of tumor cells by recombinant endostatin prevents experimental hepatic melanoma metastasis

We investigated effects of endostatin (ES) in the prometastatic microenvironment of inflammation occurring during the microvascular phase of cancer cell infiltration in the liver. We used a model of intrasplenic injection of B16 melanoma (B16M) cells leading to hepatic metastasis through vascular cell adhesion molecule-(VCAM-1)-mediated capillary arrest of cancer cells via interleukin-18 (IL-18)-dependent mechanism. We show that administration of 50 mg/kg recombinant human (rh) ES 30 min before B16M, plus repetition of same dose for 3 additional days decreased metastasis number by 60%. A single dose of rhES before B16M injection reduced hepatic microvascular retention of luciferase-transfected B16M by 40% and inhibited hepatic production of tumor necrosis factor alpha (TNF-alpha) and IL-18 and VCAM-1 expression by hepatic sinusoidal endothelia (HSE). Consistent with these data, rhES inhibited VCAM-1-dependent B16M cell adhesion to primary cultured HSE receiving B16M conditioned medium, and it abolished the HSE cell production of TNF-alpha and IL-18 induced by tumor-derived vascular endothelial cell growth factor (VEGF). rhES abrogated recombinant murine VEGF-induced tyrosine phosphorylation of KDR/flk-1 receptor in HSE cells, preventing the proinflammatory action of tumor-derived VEGF on HSE. rhES also abolished hepatic production of TNF-alpha, microvascular retention of luciferase-transfected B16M, and adhesion of B16M cells to isolated HSE cells, all of them induced in mice given 5 micro g/kg recombinant murine VEGF for 18 h. This capillary inflammation-deactivating capability constitutes a nonantiangiogenic antitumoral action of endostatin that decreases cancer cell arrest within liver microvasculature and prevents metastases promoted by proinflammatory cytokines induced by VEGF.     

Inhibition of invasion and metastasis of gastric cancer cells through snail targeting artificial microRNA interference

The zinc-finger factor Snai1 plays an important role in the down-regulation of E-cadherin expression and in the induction of epithelial-mesenchymal transition (EMT) during cancer progression. In gastric cancer tissues, we noted that Snail is abnormally high expressed and is remarkably associated with the lymph node metastasis. Using a plasmid containing newly synthesized artificial microRNA (amiRNA), we transfected gastric cancer cells to block Snail expression. Both Snail protein and mRNA levels were significantly decreased in stably transfected cells, while protein and mRNA expression of E-cadherin was up-regulated. In addition, migration and invasion potential were significantly decreased after knockdown of Snail.     

Inhibition of experimental and spontaneous pulmonary metastasis of murine RCT (+) sarcoma by beta-cyclodextrin-benzaldehyde

The effect of beta-cyclodextrin-benzaldehyde (CDBA) on pulmonary metastasis in C3H/He mice was examined. In experimental metastasis that was induced by iv injection of 1 X 10(6) RCT (+) cells, the highest inhibition was observed in the mice that were treated daily with CDBA (5 mg/day) for 1 week before tumor cell inoculation and further treated for 3 weeks after inoculation, when compared with those in other experimental groups that were given only pretreatment or posttreatment. The inhibitory effect was dose-dependent. In spontaneous metastasis that was induced by sc injection of 3 X 10(6) RCT (+) cells, the inhibition of metastasis was also observed in the mice treated with CDBA (5 mg/day) in the same manner as described above. However, the development of the primary tumor was not inhibited. CDBA-treated tumor-bearing mice showed almost as much NK activity as normal mice. Furthermore, although injection of 5-fluorouracil suppressed this activity to about 50% of that in normal mice, the combined treatment with CDBA could maintain the NK cell activity at the normal level. The results suggested that the inhibition of pulmonary metastasis might be induced by a combined effect of CDBA; that is, the direct inhibition of tumors and the maintenance of NK cell activity.     

Inhibition of cancer cell invasion and metastasis by genistein

Genistein is a small, biologically active flavonoid that is found in high amounts in soy. This important compound possesses a wide variety of biological activities, but it is best known for its ability to inhibit cancer progression. In particular, genistein has emerged as an important inhibitor of cancer metastasis. Consumption of genistein in the diet has been linked to decreased rates of metastatic cancer in a number of population-based studies. Extensive investigations have been performed to determine the molecular mechanisms underlying genistein’s antimetastatic activity, with results indicating that this small molecule has significant inhibitory activity at nearly every step of the metastatic cascade. Reports have demonstrated that, at high concentrations, genistein can inhibit several proteins involved with primary tumor growth and apoptosis, including the cyclin class of cell cycle regulators and the Akt family of proteins. At lower concentrations that are similar to those achieved through dietary consumption, genistein can inhibit the prometastatic processes of cancer cell detachment, migration, and invasion through a variety of mechanisms, including the transforming growth factor (TGF)-β signaling pathway. Several in vitro findings have been corroborated in both in vivo animal studies and in early-phase human clinical trials, demonstrating that genistein can both inhibit human cancer metastasis and also modulate markers of metastatic potential in humans, respectively. Herein, we discuss the variety of mechanisms by which genistein regulates individual steps of the metastatic cascade and highlight the potential of this natural product as a promising therapeutic inhibitor of metastasis.     

Inhibition by human recombinant tissue inhibitor of metalloproteinases of human amnion invasion and lung colonization by murine B16-F10 melanoma cells

The human tissue inhibitor of metalloproteinases (TIMP) is a glycoprotein with a molecular weight of 28,000. It appears to be ubiquitous in human mesoderm tissues and has previously been shown to be identical to the collagenase inhibitor isolated from human skin fibroblasts. TIMP inhibits type I- and IV-specific collagenases and other neutral metalloendoproteinases that may be responsible for the degradation of extracellular matrix in tumor cell metastasis. In this work we have utilized recombinant human TIMP (rTIMP) obtained by expression of its cDNA gene (Carmichael et al., Proc. Natl. Acad. Sci. USA, 83:2407, 1986). The rTIMP is shown to have similar inhibition properties as natural TIMP against human skin fibroblast collagenase. In an in vitro amnion invasion assay system, rTIMP inhibited the invasion of B16-F10 murine melanoma cells through the human amniotic membrane at an identical concentration to that reported previously for natural TIMP. The mechanism by which rTIMP inhibits amniotic membrane invasion was compared to the mechanism by which the fibronectin receptor binding peptide RGDS and the aminin receptor binding peptide YIGSR inhibit amnion invasion. RGDS and YIGSR inhibited strong binding of the tumor cells to the amniotic membrane. In contrast rTIMP did not inhibit the cell adhesion step in amnion invasion, but actually increased the number of tumor cells that were tightly bound to the amnion. Thus rTIMP appears to inhibit a later step in the amnion invasion process, following B16-F10 cell adhesion. C57BL/6 mice treated with i.p. injections of rTIMP every 12 h for 6.5 days showed a significant inhibition of metastatic lung colonization by B16-F10 murine melanoma cells. While the rTIMP inhibited the number of metastatic lung tumors formed, it had no significant effect on the size of the lung tumors. Furthermore, tumors grown s.c. in mice receiving 12-h i.p. injections of rTIMP for 6.5 days, as in the in vivo colonization assay, showed no difference in size from controls. Thus the anticolonization effect of rTIMP appears not be due to an effect on tumor growth, but on the invasion step itself. The inhibition of lung colonization in C57BL/6 mice by rTIMP is one of the first examples showing an antimetastatic effect of a selective metalloproteinase inhibitor in a mammalian animal model, and supports an essential role for metalloproteinase(s) in the extravasation and invasion of tumor cells during lung colonization by blood-borne tumor cells.     

Inhibition of breast cancer cell invasion by melatonin is mediated through regulation of the p38 mitogen-activated protein kinase signaling pathway

Introduction  

The pineal gland hormone, melatonin, has been shown by numerous studies to inhibit the proliferation of estrogen receptor α (ERα)-positive breast cancer cell lines. Here, we investigated the role of melatonin in the regulation of breast cancer cell invasion.     

Inhibitory effect of Cordyceps sinensis on experimental hepatic metastasis of melanoma by suppressing tumor cell invasion

We investigated the anti-metastatic activity of a water extract of Cordyceps sinensis (WECS) using a model of mice injected with B16-F0 mouse melanoma cells into the spleen. WECS administered intraperitoneally reduced the number of metastatic surface nodules of B16-F0 cells in the liver of C57BL/6Cr mice in a dose-dependent manner, and significantly prolonged their survival. To identify the mechanism of the anti-metastatic effect of WECS, we examined its effects on hepatocyte growth factor (HGF)-accelerated invasion of B16-F0 cells using a chemo-invasion assay in vitro. As a result, WECS reduced HGF-accelerated B16-F0 cell invasion in a concentration-dependent manner. These findings suggest that WECS exerts an anti-metastatic action, in part by inhibiting the HGF-accelerated tumor invasiveness of mouse melanoma cells.     

Inhibition of experimental metastasis and cell adhesion of B16F1 melanoma cells by inhibitors of protein kinase C.

Phorbol esters which activate protein kinase C (PKC) have been shown to enhance experimental lung metastasis. Therefore, it was reasoned that inhibitors of PKC might also modulate metastasis. We have investigated this possibility using a PKC inhibitor, MDL 27,032 [4-propyl-5(4-pyridinyl)-2(3H)-oxazolone], as well as staurosporine and H-7. Treatment of B16F1 murine melanoma cells with MDL 27,032 for 24 h in culture and subsequent i.v. injection of the cells into mice resulted in greater than 90% inhibition of lung metastasis. Inhibition of metastasis was time dependent, with 90% of maximum inhibition occurring by 8 h of incubation. The 50% inhibitory concentration (IC50) for inhibition of metastasis with MDL 27,032 was 7 microM, a value similar to that for the inhibition of B16F1 membrane-associated PKC (IC50 = 13 microM) but not cytosolic PKC (IC50 = 54 microM). B16F1 cells treated with MDL 27,032 for 24 h were less adherent than untreated cells to extracellular matrix/basement membrane proteins. Adhesion to fibrinogen and collagen IV was inhibited (IC50 = 6 microM and 48 microM, respectively) by MDL 27,032, whereas adherence to laminin and fibronectin was not affected, indicating that the drug affects specific adhesion molecules. MDL 27,032-treated cells were also found to be less adherent than untreated cells to human umbilical vein endothelial cells. The phosphorylation of an 80-kDa B16F1 cell plasma membrane protein was stimulated under conditions known to stimulate PKC activity, and MDL 27,032 inhibited this phosphorylation in a dose-dependent manner. MDL 27,032 was more potent than H-7 for the inhibition of metastasis but was significantly less potent than staurosporine. These results support the hypothesis that there is a critical role for PKC-mediated phosphorylation of cell surface adhesion receptors in metastasis.     

Inhibition of the metastasis of murine malignant melanoma by synthetic polymeric peptides containing core sequences of cell-adhesive molecules

We investigated that the antimetastatic and antiadhesive activities of peptides based on Arg-Gly-Asp adhesive signal in fibronectin could be augmented by their polymerization. Poly(Arg-Gly-Asp), which consists of a repetitive sequence of Arg-Gly-Asp, inhibited lung metastases in C57BL/6 mice more effectively than Arg-Gly-Asp tripeptide was able to do, when coinjected or separately injected with B16-BL6 cells. The adhesion of tumor cells to fibronectin was specifically inhibited by adding poly(Arg-Gly-Asp) but not unrelated peptides. In contrast, poly(Arg, Gly, Asp), in which three amino acids are randomly arranged, showed neither inhibition of lung metastases nor any adhesive ability to attach to tumor cells. The inhibitory effect of polymeric peptides containing the Arg-Gly-Asp sequence on lung metastases decreased according to the decreasing repeat units of the Arg-Gly-Asp core sequence. Polymeric peptides with Arg-Gly-Asp entrapped within the liposome membranes also caused a remarkable reduction of metastatic colonies. In a spontaneous metastasis model, multiple i.v. administrations of poly(Arg-Gly-Asp) after tumor inoculation caused the significant reduction of metastatic colonies in the lung but did not affect the growth (size) of primary tumor. We found that the polymerization (multivalency) of the Arg-Gly-Asp core sequence was able to augment the inhibition of tumor lung metastases in experimental and spontaneous metastasis models as well as the cell-adhesive property more effectively than a monovalent unit of Arg-Gly-Asp peptide.     

Suppression by cathepsin L inhibitors of the invasion of amnion membranes by murine cancer cells

Cysteine proteinases, particularly cathepsins B and L, have been strongly implicated in fostering metastasis in mice. In this work four different inhibitors of cysteine proteinases have been shown to inhibit the invasion of the human amnion by murine melanoma and mammary carcinoma cells in vitro. Two of the inhibitors are synthetic peptides [ZPhePheCHN2 (benzyloxycarbonyl-L-phenylalanyl-L-phenylalanyldiazomethane) and ZPheAlaCH2F [3-(N-benzyloxycarbonylphenylalanylamido)-DL-1-fluoro-2-butanone]] and two are thiol protease inhibitors (TPIn, TPId) isolated from the skeletal muscle of the hind limbs of normal and dystrophic mice, respectively. The inhibitors (ZPhePheCHN2, TPId), with apparent selectivity for cathepsin L, blocked invasion as effectively as inhibitors (ZPheAlaCH2F, TPIn) effective on both cathepsins. The data reveal that in these cell lines the cysteine proteinases contribute significantly to the invasive capacity of the cells, but to a lesser extent than do the metalloproteinases. We suggest that the cysteine proteinases facilitate the action of metalloproteinases (collagenase, gelatinase, and stromelysin), possibly by activating them, by inactivating the tissue inhibitor of metalloproteinases, and/or by making basement membrane matrix more accessible.     

Inhibition of experimental tumor metastasis by selective activation of natural killer cells

The antitumor activity generated by selective activation of natural killer (NK) cells was studied in vitro and in vivo. Unlike Corynebacterium parvum CN6134, which activated both NK cells and macrophages, periodate-oxidized C. parvum CN6134 lost the ability to activate macrophages but retained almost all the NK-stimulating capacity of the untreated bacterium. The "inactive" C. parvum strain CN5888 also induced a modest, but selective, activation of NK cells. The enhanced NK cell-mediated cytotoxicity was expressed against YAC-1 lymphoma, UV-2237 fibrosarcoma, and B16 melanoma target cells in vitro and was manifested in vivo by increased destruction of circulating tumor cells and the inhibition of hematogenous tumor metastasis. Periodate-treated C. parvum was as effective in inhibiting the formation of B16 melanoma pulmonary metastases as was untreated C. parvum. In both cases, the inhibiting effect corresponded closely with the kinetics of NK cell activation.     

Cloning efficiency of human melanoma cells is modulated after invasion through a reconstituted basement membrane

Three human malignant melanoma cell strains (C8146A, C8146C, and C83-2CY), three established human melanoma cell lines (A375P, A375M, and C8161), and one selected human melanoma subline (A375P-5) were studied to determine if invasion through a reconstituted basement membrane-coated filter (RBMF), which selects the most aggressively invasive cells, would also modulate the cloning efficiency of these cells in soft agar. With the use of the Membrane Invasion Culture System (MICS), all cell strains tested showed a significant increase in cloning efficiency (1.05-9.3-fold) following transit through the RBMF when compared to unmanipulated populations. The established cell lines (A375P, A375M, and C8161) and the A375P-5 subline showed either a decrease or unaltered status in cloning efficiency after invasion. However, all cells demonstrated a consistent decrease in clonogenicity following transit through an uncoated filter compared with RBMFs, thus suggesting the influence of the extracellular matrix on tumor cell clonogenic properties. In general, the established cell lines were more clonogenic before invasion of the RBMF compared with the cell strains, and no correlation was found between clonogenic potential and invasive or metastatic capability. These data may provide important insight into the underlying mechanisms of tumor cell invasion and the subsequent formation and dissemination of metastases in vivo.     

Inhibition of tumor growth and vasculogenic mimicry by curcumin through down-regulation of the EphA2/PI3K/MMP pathway in a murine choroidal melanoma model

This study aims to investigate the underlying mechanism by which curcumin inhibits tumor growth and reduces vasculogenic mimicry (VM) in a murine choroidal melanoma model. Sixty mice were given subretinal injection with B16F10 cells and divided into a treatment and a control group. Curcumin was administered to the treatment group once a day at a dose of 100 mg/kg for 18 days starting at d3 (the day of inoculation is designated as d0); an equivalent volume of poloxamer-F68 was administered to the control group. Immunohistochemical and histochemical double staining were ued to detect the different blood supply patterns. The amounts of epithelial cell kinase (EphA2), phosphatidylinositol-3-kinase (PI3K), and matrixmetalloproteinase-2 and -9 (MMP-2, MMP-9) proteins expressed in the tumor tissue were analyzed using immunohistochemical staining; mRNA levels were measured using real-time PCR analysis. Results indicate that the tumor volume is reduced (P=0.000) and that the numbers of VM (P=0.000), mosaic vessels (P=0.031), and endothelium-dependent vessels (P=0.000) are significantly decreased by curcumin (P=0.001). The expression levels of EphA2, PI3K, MMP-2, and -9 are also lower in the treatment group than in the control group (P=0.001); similarly, mRNA levels in the treatment group are lower than those in the control group (P=0.000). In conclusion, curcumin has the ability to inhibit the growth of engrafted melanoma VM channels through the regulation of vasculogenic factors that could be related to the down-regulation of the EphA2/PI3K/MMPs signaling pathway. Thus, curcumin has the potential of being a clinical inhibitor of VM of choroidal melanoma.