Inhibition of fibrinolysis by a synthetic urokinase inhibitor enhances lung colonization of metastatic murine mammary tumor cells

We have investigated the role of coagulation and fibrinolysis during the metastatic lung colonization of F3II mouse mammary carcinoma cells. The selective synthetic urokinase inhibitor B623 significantly enhanced lung colonization and blocked the antimetastatic effect of heparin when administered i.p. during the first stages of metastasis formation. In B623-treated mice the overall activity of the fibrinolytic system was reduced and circulating urokinase was specifically inhibited by this agent. In vitro studies demonstrated that B623 induces the aggregation of F3II cells in the presence of mouse plasma and facilitates the entrapment of tumor cells in a fibrin gel matrix. Our data suggest that imbalances of fibrin deposition and removal may dramatically influence metastatic lung colonization.     

Adhesion of metastatic and non-metastatic carcinoma cells to glass surfaces

Adhesion in vitro is described in cells from a tumor pair originating from a single methylcholanthrene-induced mouse carcinoma. One member of this tumor pair shows a high incidence of metastases while the other does not metastasize. Cells from the non-metastasizing carcinoma were found to form close and focal contacts with a glass substrate consecutively, as do normal mouse kidney epithelial cells. Cells from the metastasizing carcinoma, however, had only limited areas of close contact and generally failed to form focal contacts. It is suggested that this alteration in cell-substrate adhesion contributes to the release and mobility of metastatic cells.     

Lewis肺癌细胞趋化因子受体CXCR4表达的研究

目的:观察趋化因子受体CXCR4在Lewis肺癌细胞和肿瘤组织中的表达,为探讨CXCR4与肿瘤转移的关系提供研究基础。方法:体外培养Lewis肺癌细胞,并建立Lewis肺癌细胞皮下种植瘤模型与自发性转移模型,采用RT-PCR和蛋白质印迹法分析培养细胞和肿瘤组织中CXCR4 mRNA及蛋白质表达水平;应用免疫组化方法分析Lewis肺癌原发瘤和转移瘤中CXCR4表达,并通过迁移实验观察CXCR4特异性配体SDF-1α对Lewis肺癌细胞的趋化作用。结果:培养Lewis肺癌细胞及肿瘤组织中均功能性地表达趋化因子受体CXCR4,且其表达水平与细胞所处氧环境有关;低氧能够促使Lewis肺癌细胞CXCR4蛋白表达增高,与对照组相比差异有统计学意义,t=4.051,P=0.009。12h细胞趋化运动实验显示,CXCR4特异性配体SDF-1α可剂量依赖性诱导Lewis肺癌细胞的趋化运动,在低氧条件下,同对照组相比SDF-1α质量浓度为25ng/mL时开始促进细胞迁移,t=3.053,P=0.031;SDF-1α质量浓度为50ng/mL时可明显提高细胞迁移率,t=4.521,P=0.004。结论:Lewis肺癌细胞功能性表达趋化因子受体CX-CR4,可能与肿瘤细胞迁移和转移有关。     

Effect of prostaglandin E2-producing nonmetastatic Lewis lung carcinoma cells on the migration of prostaglandin E2-responsive metastatic Lewis lung carcinoma cells

The role of prostaglandin E2 (PGE2) in directly stimulating metastatic spread by Lewis lung carcinoma (LLC) cells was examined with the use of an in vitro migration model for tumor dissemination. The extent to which cloned metastatic and nonmetastatic LLC cells migrated out of glass capillary tubes in vitro reflected their capacity to form pulmonary metastases in vivo. The addition of PGE2 to metastatic LLC cells further stimulated their migration. Other cyclooxygenase products, besides PGE2, did not stimulate the migration of metastatic LLC cells. Nonmetastatic LLC cells did not migrate out of capillary tubes, even in the presence of exogenous PGE2. The amount of PGE2 secreted by cloned LLC cells was quantitated by a radioimmunoassay. Nonmetastatic LLC cells secreted more PGE2 than did the metastatic LLC cells. When the nonmetastatic LLC cells were either mixed with or placed adjacent to cloned metastatic LLC cells, the migration by the metastatic LLC cells was stimulated. The migration-stimulatory capacity of the nonmetastatic LLC cells was minimized in the presence of indomethacin, a prostaglandin synthesis inhibitor. Studies were conducted to relate these in vitro results to tumor metastasis in vivo. Injection of a mixture of metastatic and nonmetastatic LLC cells into mice s.c. resulted in a greater number of lung metastases than did injection of metastatic cells alone. This increase in metastasis formation was prevented by indomethacin. Formation of pulmonary metastases was also augmented when irradiated nonmetastatic LLC cells were injected into metastatic LLC-bearing mice. The results of our studies suggest that nonmetastatic LLC cells, by producing PGE2, can augment in vitro migration and in vivo dissemination of metastatic LLC cells. Thus, the response of tumor cells to PGE2, rather than simply their production of PGE2, appears to be important in regulating tumor dissemination.     

MHC imbalance and metastatic spread in Lewis lung carcinoma clones

Imbalance in the K^b and D^b region encoded molecules is observed in Lewis lung carcinoma clones. The uncloned metastatic population and the D122 high-metastatic clone show no expression of H-2K^b products, while the non-metastatic A9 clone expresses K^b products. Twenty-nine new subclones of 3LL and A9 were analyzed for D-end and K-end membrane expression, primary growth rate and metastatic spread. We show that the imbalance in H-2K^b to H-2D^b is correlated with metastatic properties of a given clone, but local tumor growth is not. A “low K^b/low D⁹” phenotype is nonmetastatic as is a “high K^b/high D^b” phenotype; a “low K^b/high D^b” is highly metastatic and a “medium K^b/high D^b” is moderately metastatic. We find support for this notion of imbalance in experiments on MHC modulation by interferon and retinoic acid. Interferon increases both K^b and D^b expression of A9 and D122 clones yet the net increase of D^b was greater than K^b. This was associated with an increase in metastasis formation. Retinoic acid increases the expression of the D^b gene product on the nonmetastatic A9, clone, without apparent changes in K^b expression. This treatment shifts the A9 to a high-metastatic phenotype. The significance of this imbalance to the tumor — host relationship is discussed.     

Inhibition of metastasis of lewis lung carcinoma by urinary trypsin inhibitor in experimental and spontaneous metastasis models

A purified human urinary trypsin inhibitor (UTI) and its related synthetic peptides were examined to determine whether they could inhibit production of experimental and spontaneous lung metastases by murine Lewis lung carcinoma (3LL) cells. Three peptides, peptide 1, peptide 2 and peptide 3, representing the amino acid sequences within the UTI molecule, were synthesized. UTI and peptide 2 inhibited human leukocyte elastase (HLE). UTI and peptide 3 specifically inhibited human and murine plasmin activity. Peptide 1 had essentially no inhibitory activity. In an in vivo spontaneous metastasis model, multiple s.c. injections of UTI or peptide 3 for 7 days immediately after s.c. tumor cell inoculation significantly inhibited the formation of lung metastasis in CS7BL/6 mice in a dose-dependent manner. UTI reduced lung tumor colonization more effectively than peptide 3. Peptides 1 and 2, however, did not affect the formation of lung metastasis. Inhibition of lung metastasis was not due to direct anti-tumor effects of UTI and peptide 3. In an in vivo experimental metastasis assay, multiple s.c. injections of UTI for 7 days after i.v. tumor cell inoculation inhibited metastatic lung tumor colonization, while peptide 3 did not affect metastasis. Peptides 1 and 2 did not affect the formation of lung metastasis. When examined with an in vitro assay system using a modified Boyden chamber, UTI and peptide 3 suppressed the invasion of tumor cells through Matrigel. UTI and peptide 3 inhibited neither cell proliferation nor the binding of tumor cells to Matrigel and showed no significant suppression of chemotactic migration of tumor cells to fibronectin. Our results suggest that UTI efficiently regulates the mechanism involved in not only the entry into vascular circulation of tumor cells (intravasation, though, at least in part, inhibition of the proteolytic enzyme plasmin) but also the extravasation step of the metastatic process.     

Isolation of urinary trypsin inhibitor-like inhibitor from human lung cancer tissue

In the present study, a trypsin inhibitor was first extracted from lung cancer tissue and purified by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. A final yield of 20 to 60 micrograms of inhibitor with a specific activity of 2040 units/mg of protein was obtained from 1 g of original lung cancer tissue. This inhibitor inhibited trypsin strongly, plasma kallikrein weakly, and plasmin more weakly, and its molecular weight was approximately 43,000 to 45,000 as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Its antigenicity was confirmed to be quite the same as that of human urinary trypsin inhibitor by double immunodiffusion, immunoelectrophoresis, and neutralization with anti-urinary trypsin inhibitor rabbit immunoglobulin.     

Enhanced metastatic potential of cloned low-metastatic Lewis lung carcinoma cells treated in vitro with dimethyl sulfoxide

Treatment of low-metastatic Lewis lung carcinoma cells (P-29) with dimethyl sulfoxide in vitro enhanced their lung-colonizing ability. The concentration of dimethyl sulfoxide used delayed the in vitro growth of P-29 cells but was not cytotoxic. The arrest and retention in the lung of untreated and dimethyl sulfoxide-treated P-29 cells labeled with 5-[125I]iodo-2'-deoxyuridine after injecting them into a tail vein of syngeneic mice were examined. Dimethyl sulfoxide-treated P-29 cells were trapped in the lung more than untreated cells and were cleared from the lungs more slowly than untreated cells. Treatment of P-29 cells with dimethyl sulfoxide resulted in the increase in their homotypic aggregation and adhesion to plastic culture dishes, monolayers of endothelial cells, and a subendothelial extracellular matrix. This treatment also increased significantly their activities of degradative enzymes, such as glycosidases and cathepsin B, and their production of plasminogen activator. These results indicate that the enhanced lung-colonizing ability of P-29 cells treated with dimethyl sulfoxide is due to the increase in adhesiveness, resulting in arrest and retention of the cells in the lung of the host and in the increase in their degradative enzyme activities. The enhancing effect of dimethyl sulfoxide on the lung-colonizing ability of P-29 cells was found to be reversible.     

Collagen degradation by metastatic variants of Lewis lung carcinoma: cooperation between tumor cells and macrophages

Interactions between cancer cells and host macrophages might have important regulatory roles in controlling the expression of the metastatic phenotype, particularly by regulating the production of proteases necessary for tissue invasion. To investigate that possibility, mouse macrophages and Lewis lung carcinoma (LLC) cells from four clonal subpopulations with either low or high metastatic ability were cultured on [14C]collagen (type l)-coated plates. They did not degrade collagen when they were cultured independently on that substrate, but they were induced to do so when macrophages and cancer cells were cultured together. An increased production of neutral collagenase and other neutral protease activities was observed simultaneously. The degree of stimulation of collagen degradation varied according to the cancer cell subpopulation present in the cocultures. For a given LLC cell subpopulation, similar degrees of stimulation of collagen degradation were achieved with either bone marrow-derived or resident peritoneal macrophages, either syngeneic (from C57BL/6 mice) or allogeneic; lower stimulations were obtained with thioglycolate-elicited peritoneal macrophages. Macrophage-conditioned culture media could be substituted for living macrophages to stimulate collagen degradation or collagenase secretion by LLC cells, but LLC cell-conditioned media did not stimulate collagen degradation by macrophages. This suggests that, in the cocultures, collagen degradation is achieved mainly by the cancer cells, not by the macrophages, and that it is induced by a soluble factor, a monokine, produced by the macrophages. That factor might be identical to a recently identified rabbit monokine that stimulates fibroblasts or synovial cells to degrade collagen and proteoglycan and to activate plasminogen, because rabbit macrophage-conditioned media containing that monokine also stimulated collagen degradation by LLC cells.     

Modulation of metastatic potential by cell surface urokinase of murine melanoma cells

We have carried out enzymatic, immunofluorescence, and surface iodination studies which show that B16 melanoma cells express the single chain form of the urokinase type plasminogen activator (uPA) on their cell surface, and that these cells are capable of plasminogen-dependent fibronectin degradation. The significance of the expression of surface single-chain uPA and uPA activity to the metastatic process was examined by preincubating melanoma cells with uPA modulating agents followed by i.v. injection of the cells into mice and enumeration of pulmonary nodules 17 days later. B16 cells that had been pretreated with anti-uPA immunoglobulins that were inhibitory to uPA activity invariably showed significantly decreased numbers of metastases compared to controls. On the contrary, pretreatment with plasmin, which is not only the product of the uPA catalyzed reaction but is also able to convert single-chain uPA to uPA, significantly increased the numbers of metastases. Control treatments, which included normal rabbit and mouse immunoglobulins, monovalent noninhibitory anti-uPA Fab fragments, and various monoclonal and polyclonal antibodies directed against other B16 cell surface antigens, did not affect the metastatic potential of the cells. Divalent inhibitory anti-uPA F(ab)2 fragments, on the contrary, inhibited metastasis as efficiently as intact IgG. The results support the hypothesis that proteolysis of extracellular matrix components by cell surface-localized uPA may be a critical step during the process of tumor cell invasion and metastasis.     

Expression of coxsackie and adenovirus receptor reduces the lung metastatic potential of murine tumor cells

The coxsackie and adenovirus receptor (CAR) is involved in the epithelial cell tight junction, the downregulated expression of which is observed in different cancer types. In the present study, we examined CAR's role in tumor metastasis using a B16 melanoma and CT26 colon adenocarcinoma model of experimental metastasis. In lung metastasis, the colony number of B16 cells stably expressing CAR (B16CAR) was significantly lower than that of the control CAR-negative B16 cells. B16 and CT26 cells transiently expressing CAR, which were transduced with adenovirus (Ad) vector expressing CAR, also reduced lung metastasis, suggesting that CAR plays a role in the early stage of metastasis. CAR expression significantly decreased the accumulation of B16 cells in the lung after i.v. injection and the migration in vitro. CAR expression reduced expression of alpha(v), alpha(4), beta(3) and beta(1) integrin, which play important roles in attachment to cells or basement membrane. Thus, CAR expression likely acts as a metastatic suppressor.     

Expression of tumor antigen correlated with metastatic potential of Lewis lung carcinoma and B16 melanoma clones in mice

Expression of a tumor-associated antigen, recognized by a monoclonal antibody (MoAb 135-13C) to lung carcinoma cells, has been studied in cloned Lewis lung carcinoma (3LL) and in B16 melanoma (F1 and F10) tumor lines endowed with different metastatic potentials. MoAb 135-13C recognizes a protein complex (tumor-specific Mr 180,000 protein) that appears on the cell surface of several murine lung carcinomas but is not detected on normal cells in culture. Standard metastatic variants of B16 melanoma (F1 and F10) and two variant sublines of 3LL (M1087 and BM21548) together with the parental line of 3LL have been used for these experiments. The two cloned variant lines derived from 3LL have been shown to retain high (M1087) and low (BM21548) metastatic phenotypes during in vivo passaging. We found that all three cell lines of 3LL bind monoclonal antibody specifically, but one cell variant with higher metastatic potential shows a higher capacity to bind MoAb 135-13C than did the other variant. Similarly we found that B16 F10 cells bind higher amounts of MoAb 135-13C than did B16 F1 cells. In addition the analysis of the amounts of MoAb 135-13C bound to the cell surface of several other in vitro and in vivo tumor lines with different metastatic capacity demonstrates that all tumor lines which express high ability to colonize to the lung also express, on the cell surface, higher amounts of tumor-specific Mr 180,000 protein. The sodium dodecyl sulfate-polyacrylamide gel electrophoresis autoradiograms of immunoprecipitates from cell lysates of 3LL and B16 tumor lines demonstrate that MoAb 135-13C specifically precipitated three proteins banding at molecular weights of 204,000, 134,000, and 116,000. We conclude that MoAb 135-13C recognizes a surface protein complex which is present in higher amounts in 3LL and B16 cells which possess higher capacity to metastasize to the lung.     

Metastatic heterogeneity of cells from Lewis lung carcinoma

To allow investigations of the role of tumor cell proteases in invasion and metastasis, an attempt was made to obtain well-defined homogeneous populations of Lewis Lung carcinoma cells differing widely in their metastatic potential. From a single Lewis lung carcinoma, a parental line of cells was established and subsequently cloned so as to provide 18 clonal tumor cell lines. These clones differed in their ability to produce spontaneous, macroscopically visible metastases in the lung after i.m. inoculation into syngeneic C57BL/6 mice. Several of them were less metastatic than the parental line. The parental line expressed a metastatic behavior close to that of the high-metastatic cell subpopulations that it contained. There was, within certain limits, a good correlation between the potential for spontaneous lung metastases arising from a primary tumor and that for "artificial" lung colonies obtained after i.v. injection of the Lewis lung carcinoma cells. Although positively correlated with the growth rate of the tumor cells, the metastatic ability of the clones could not be considered as a mere reflection of the proliferation rates of the cells constituting the primary tumors. Differences in metastatic behavior observed among clones persisted in several cases after the cells had been maintained in culture for prolonged periods. However, this stability of the clones in vitro was not absolute. Indeed, some subclones isolated from the low-metastatic clone H122 displayed metastatic abilities which were lower than that of the parent clone. Furthermore, a significant increase in metastatic potential was once observed after a prolonged culture period of that same clone, H122. Thus, new metastatic phenotypes can emerged under in vitro culture conditions. However, the relative rarity of this event suggests that some metastatic heterogeneity already preexisted in vivo among the tumor cells.     

THE CHARACTERISTICS OF LAMININ RECEPTOR ISOLATED FROM MURINE LUNG LEWIS CARCINOMA

Laminin receptor(LN-R, Mr=70,000) isolated from murine Lewis lung carcinoma was shown to be a single band on SDS-PAGE (Sodium Dodecyl Sulfate-Polyacrylamide gel electrophoresis) after reduction. The present study Indicates that LN-R was a kind of glycoproteins by periodic add Schiff staining. It's pI was 4. 93. LN-R preperation was associated with phospholipid and neutral glycolipid by TLC (thin layer chromatography) or HPTLC (high performance thin layer chromtography) of chloroform-methanol extracts of LN-R, respectively, but without acidic glycolipid. Binding of LN-R with its ligand, Lamlnin, depended on the presence of Ca , Mg . LN-R may bind to actin.     

PP1蛋白在转移及非转移性恶性黑色素瘤中的表达

目的：研究蛋白磷酸酶-1（PP1）对恶性黑色素瘤转移的作用。方法：采用Western blot检测PP1蛋白在非转移性恶性黑色素瘤细胞系-HMCB及转移性恶性黑色素瘤细胞系WM266-4中的表达。用免疫组化的方法检测32例未转移恶性黑色素瘤及20例转移性恶性黑色素瘤标本中PP1的表达。结果：本研究证明非转移性恶性黑色素瘤细胞及组织中PP1蛋白表达明显高于在转移性恶性黑色素瘤细胞及组织中的表达,差异有统计学意义（P〈0.05）。结论：PP1在转移及未转移恶性黑色素瘤细胞及组织中的差异表达为恶性黑色素瘤转移机制方面的研究开辟新的方向。     

PP1蛋白在转移及非转移性恶性黑色素瘤中的表达

目的 研究蛋白磷酸酶-1(PP1)对恶性黑色素瘤转移的作用.方法 采用Western blot检测PP1蛋白在非转移性恶性黑色素瘤细胞系-HMCB及转移性恶性黑色素瘤细胞系WM266-4中的表达.用免疫组化的方法检测32例未转移恶性黑色素瘤及20例转移性恶性黑色素瘤标本中PP1的表达.结果 本研究证明非转移性恶性黑色素瘤细胞及组织中PP1蛋白表达明显高于在转移性恶性黑色素瘤细胞及组织中的表达,差异有统计学意义(P<0.05).结论 PP1在转移及未转移恶性黑色素瘤细胞及组织中的差异表达为恶性黑色素瘤转移机制方面的研究开辟新的方向.     

CD44 expression in metastatic and non-metastatic non-small cell lung cancers

CD44 is an integral membrane glycoprotein that functions as a receptor for the extracellular matrix glycan, hyaluronan. It exists as standard (CD44s) and variant (CD44v) isoforms which have been shown to be associated with metastasis in a range of tumors. Both CD44s and CD44v are found in normal respiratory epithelium and in non small cell lung cancers (NSCLCs). However, there isn't much information regarding their role in the metastatic process of NSCLCs. We examined the expression of CD44s in 30 NSCLCs using immunohistochemical techniques. Ten were non-metastatic (N0, M0), 5 were metastases to lymph nodes (N1, M0) and 15 were excised brain metastases. We found CD44s positivity in all non-metastatic tumors with varying degrees of expression, whereas 10 out of 15 metastatic tumors in brain and 3 out of 5 lymph node metastases were negative for CD44s. There was a statistically significant inverse relation between the CD44s expression and metastatic potential (P < 0.05). Our results support the hypothesis that diminished or lack of CD44s is the functional equivalent of CD44v which may be an accompaniment of enhanced metastatic potential. The value of CD44 in predicting metastatic behavior of NSCLCs remains to be established in a larger series.     

Dietary supplementation with curcumin enhances metastatic growth of Lewis lung carcinoma in mice

Our study investigated the effects of dietary supplementation with curcumin [(1E,6E)‐1,7‐bis(4‐hydroxy‐3‐methoxyphenyl)‐1,6‐heptadiene‐3,5‐dione] on spontaneous metastasis of Lewis lung carcinoma (LLC) in C57BL/6 mice. Mice were fed with the AIN93G control diet or with the diet supplemented with 2 or 4% curcumin for 5 weeks at which time they were injected subcutaneously with 2.5 × 10⁵ viable LLC cells. The subcutaneous primary tumor was surgically removed when it reached ～ 8 mm in diameter, and the experiment was terminated 10 days after the surgery. There was no difference in pulmonary metastatic yield among the groups. Curcumin supplementation at either dietary level did not significantly increase the size of metastatic tumors; however, the combined data from both curcumin groups showed that curcumin treatment increased metastatic tumor cross‐sectional area by 46% (p < 0.05) and volume by 70% (p < 0.05) compared to the controls. Curcumin supplementation increased plasma concentrations of angiogenic factors angiogenin (p < 0.05), basic fibroblast growth factor (p < 0.05) and vascular endothelial growth factor (p < 0.05), as well as inflammatory cytokines interleukin‐1β (p < 0.05) and monocyte chemotactic protein‐1 (p < 0.05), compared to the controls. These results demonstrate that curcumin does not prevent metastasis and indicate that it can enhance metastatic growth of LLC in mice, perhaps through upregulation of angiogenesis and inflammation.