FGF2 binding, signaling, and angiogenesis are modulated by heparanase in metastatic melanoma cells.

Heparanase (HPSE) and fibroblast growth factor-2 (FGF2) are critical regulators of melanoma angiogenesis and metastasis. Elevated HPSE expression contributes to melanoma progression; however, further augmentation of HPSE presence can inhibit tumorigenicity. HPSE enzymatically cleaves heparan sulfate glycosaminoglycan chains (HS) from proteoglycans. HS act as both low-affinity FGF2 receptors and coreceptors in the formation of high-affinity FGF2 receptors. We have investigated HPSE's ability to modulate FGF2 activity through HS remodeling. Extensive HPSE degradation of human metastatic melanoma cells (70W) inhibited FGF2 binding. Unexpectedly, treatment of 70W cells with low HPSE concentrations enhanced FGF2 binding. In addition, HPSE-unexposed cells did not phosphorylate extracellular signal-related kinase (ERK) or focal adhesion kinase (FAK) in response to FGF2. Conversely, in cells treated with HPSE, FGF2 stimulated ERK and FAK phosphorylation. Secondly, the presence of soluble HPSE-degraded HS enhanced FGF2 binding and ERK phosphorylation at low HS concentrations. Higher concentrations of soluble HS inhibited FGF2 binding, but FGF2 signaling through ERK remained enhanced. Soluble HS were unable to support FGF2-stimulated FAK phosphorylation irrespective of HPSE treatment. Finally, cell exposure to HPSE or to HPSE-degraded HS modulated FGF2-induced angiogenesis in melanoma. In conclusion, these effects suggest relevant mechanisms for the HPSE modulation of melanoma growth factor responsiveness and tumorigenicity.

Comparison of the acute response to meals enriched with cis- or trans-fatty acids on glucose and lipids in overweight individuals with differing FABP2 genotypes.

Trans-fatty acids have been implicated as a risk factor for cardiovascular disease and diabetes. In addition, a polymorphism at codon 54 (Ala54Thr) in the fatty acid-binding protein 2 (FABP2) gene has been suggested to modify an interaction between dietary fat and insulin sensitivity. We examined the postprandial metabolic profiles after meals enriched with C18:1trans- relative to a similar meal with C18:1cis-fatty acid in individuals who were either FABP2 Ala54 homozygotes or Thr54 carriers. Moderately overweight men and women ate 2 breakfast test meals, separated by 1 week, each providing 40% of their daily energy requirement and containing 50% of energy as fat. In one meal, 10% of energy was from C18:1trans, and in the other meal, the C18:1trans was replaced with C18:1cis. Metabolic parameters were assessed during an 8-hour period. Insulin and C-peptide levels increased more after the C18:1trans meal, and this was associated with a greater fall in free fatty acids. Postprandial glucose levels and oxidation of fatty acids and carbohydrate were not different between the 2 test meals. The Thr54 allele for FABP2 increased the rise in postprandial glucose but not triacylglycerols. Fractional triacylglycerol synthetic rates were higher after consumption of the C18:1trans meal relative to the C18:1cis meal only in Thr54 carriers. These data show that a single meal enriched with C18:1trans-fatty acids can significantly increase insulin resistance, and that in the presence of the FABP2 Thr54 allele, may contribute to increased partitioning of glucose to triacylglycerols and insulin resistance.

Role of omega-3 polyunsaturated fatty acids on cyclooxygenase-2 metabolism in brain-metastatic melanoma.

Cyclooxygenase-2 (COX-2) is important in the progression of epithelial tumors. Evidence indicates that omega-6 PUFAs such as arachidonic acid (AA) promote the growth of tumor cells; however, omega-3 fatty acids [eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA)] inhibit tumor cell proliferation. We investigated the effects of omega-3 PUFA on the expression and function of COX-2 in 70W, a human melanoma cell line that metastasizes to the brain in nude mice. We show that 1) tumor necrosis factor-alpha upregulates the expression of both COX-2 mRNA and prostaglandin E2 (PGE2) production, and 2) omega-3 and omega-6 PUFA regulate COX-2 mRNA expression and PGE2 production. AA increased COX-2 mRNA expression and prostaglandin production in omega-6-stimulated 70W cells. Conversely, COX-2 mRNA expression decreased in cells incubated with EPA or DHA. AA increased Matrigel invasion 2.4-fold, whereas EPA or DHA did not. Additionally, PGE2 increased in vitro invasion 2.5-fold, whereas exposure to PGE3 significantly decreased invasion. Our results demonstrate that incubation of 70W cells with either AA or PGE2 increased invasiveness, whereas incubation with EPA or DHA downregulated both COX-2 mRNA and protein expression, with a subsequent decrease in Matrigel invasion. Taken together, these results indicate that omega-3 PUFA regulate COX-2-mediated invasion in brain-metastatic melanoma.

Brain metastases in melanoma: roles of neurotrophins.

Brain metastasis, which occurs in 20% to 40% of all cancer patients, is an important cause of neoplastic morbidity and mortality. Successful invasion into the brain by tumor cells must include attachment to microvessel endothelial cells, penetration through the blood-brain barrier, and, of relevance, a response to brain survival and growth factors. Neurotrophins (NTs) are important in brain-invasive steps. Human melanoma cell lines express low-affinity NT receptor p75NTR in relation to their brain-metastatic propensity with their invasive properties being regulated by NGF, or nerve growth factor, the prototypic NT. They also express functional TrkC, the putative receptor for the invasion-promoting NT-3. In brain-metastatic melanoma cells, NTs promote invasion by enhancing the production of extracellular matrix (ECM)-degradative enzymes such as heparanase, an enzyme capable of locally destroying both ECM and the basement membrane of the blood-brain barrier. Heparanase is an endo-beta-d-glucuronidase that cleaves heparan sulfate (HS) chains of ECM HS proteoglycans, and it is a unique metastatic determinant because it is the dominant mammalian HS degradative enzyme. Brain-metastatic melanoma cells also produce autocrine/paracrine factors that influence their growth, invasion, and survival in the brain. Synthesis of these factors may serve to regulate NT production by brain cells adjacent to the neoplastic invasion front, such as astrocytes. Increased NT levels have been observed in tumor-adjacent tissues at the invasion front of human brain melanoma. Additionally, astrocytes may contribute to the brain-metastatic specificity of melanoma cells by producing NT-regulated heparanase. Trophic, autocrine, and paracrine growth factors may therefore determine whether metastatic cells can successfully invade, colonize, and grow in the CNS.

Brain-metastatic melanoma: a neurotrophic perspective.

The brain is a unique microenvironment enclosed by the skull and maintaining a highly regulated vascular transport barrier. To metastasize to the brain, malignant tumor cells must attach to microvessel endothelial cells, invade the blood-brain barrier (BBB), and respond to brain survival and growth factors. Neurotrophins (NT) are important in brain invasion because they stimulate this process. In brain-metastatic melanoma cells, NT can promote invasion by enhancing the production of extracellular matrixdegradative enzymes such as heparanase, an enzyme capable of locally destroying both the extracellular matrix and the basement membrane of the BBB. We have examined human and murine melanoma cell lines exhibiting varying abilities to form brain metastases, and have found that they express low-affinity neurotrophin receptor p75NTR in relation to their brain-metastatic potentials. They do not, however, express trkA, the gene encoding the tyrosine kinase receptor TrkA, the high-affinity receptor for nerve growth factor (NGF), the prototypic NT. Presence of functional TrkC, the putative receptor for the invasion-promoting neurotrophin NT-3, was also expressed in these cells. Brain-metastatic melanoma cells can also produce autocrine factors and inhibitors that influence their growth, invasion, and survival in the brain. Synthesis of these factors may influence NT production by brain cells adjacent to the neoplastic invasion front, such as oligodendrocytes and astrocytes. In brain biopsies, we observed increased amounts of NGF and NT-3 in tumor-adjacent tissues at the invasion front of human melanoma tumors. Additionally, we found that astrocytes contribute to the brain-metastatic specificity of melanoma cells by producing NT-regulated heparanase. Trophic, autocrine, and paracrine growth factors may therefore determine whether metastatic cells can successfully invade, colonize, and grow in the central nervous system (CNS).

Type-1 transforming growth factor-beta differentially modulates tumoricidal activity of murine peritoneal macrophages against metastatic variants of the B16 murine melanoma.

Transforming growth factor-beta 1 (TGF-beta 1) renders mouse peritoneal macrophages tumoricidal against metastatic variants of the B16 mouse melanoma in vitro. Both direct cytotoxicity and indirect cytotoxicity were observed. A subthreshold concentration (10 U/ml) of recombinant murine interferon-gamma (rMuIFN-gamma) enhanced the direct tumoricidal activity of TGF-beta 1-activated macrophages from 29% to 88% but did not change their indirect tumoricidal profile. Data obtained from macrophages preincubated with either TGF-beta 1 or rMuIFN-gamma showed that TGF-b1 can initiate tumoricidal activity better than rMuIFN-gamma. These effects were plasma-membrane mediated because targeting macrophages with liposomal TGF-beta 1 was ineffective. The order of tumoricidal susceptibility of the B16 melanoma lines to activated macrophages was B16F1 > B16F10 > B16BL6, in inverse order of metastatic potential.

A 9-mo randomized clinical trial comparing fat-substituted and fat-reduced diets in healthy obese men: the Ole Study.

BACKGROUND: Dietary fat has been implicated as a risk factor for cardiovascular disease and obesity. OBJECTIVE: We evaluated the effect on body weight, body fat, lipids, glucose, and insulin of replacing dietary fat with olestra in moderately obese men. DESIGN: Forty-five healthy overweight men were randomly assigned to 1 of 3 diets: control diet (33% fat), fat-reduced diet (25% fat), or fat-substituted diet (one-third of dietary fat replaced by olestra to achieve a diet containing 25% metabolizable fat). Body fat was measured by dual-energy X-ray absorptiometry and visceral and subcutaneous abdominal fat by computed tomography. RESULTS: Thirty-six men completed the 9-mo study. Body weight and body fat in the fat-substituted group declined by a mean (+/- SEM) of 6.27 +/- 1.66 and 5.85 +/- 1.34 kg, respectively, over 9 mo compared with 3.8 +/- 1.34 and 3.45 +/- 1.0 kg in the control group and 1.79 +/- 0.81 and 1.68 +/- 0.75 kg in the fat-reduced diet group. At 9 mo, the mean difference in body fat between the fat-reduced and fat-substituted groups was -4.19 +/- 1.19 kg (95% CI: -6.57, -1.81), that between the control and fat-substituted groups was -2.55 +/- 1.21 kg (-0.13, -4.97), and that between the control and fat-reduced groups was 1.63 +/- 1.18 kg (3.96, -0.70). The men eating the fat-reduced diet asked for almost no extra foods, in contrast with the significantly higher requests (P < 0.05) from both of the other 2 groups. CONCLUSION: Replacement of dietary fat with olestra reduces body weight and total body fat when compared with a 25%-fat diet or a control diet containing 33% fat.

Effects of diets enriched in saturated (palmitic), monounsaturated (oleic), or trans (elaidic) fatty acids on insulin sensitivity and substrate oxidation in healthy adults.

OBJECTIVE: Diets high in total and saturated fat are associated with insulin resistance. This study examined the effects of feeding monounsaturated, saturated, and trans fatty acids on insulin action in healthy adults. RESEARCH DESIGN AND METHODS: A randomized, double-blind, crossover study was conducted comparing three controlled 4-week diets (57% carbohydrate, 28% fat, and 15% protein) enriched with different fatty acids in 25 healthy men and women. The monounsaturated fat diet (M) had 9% of energy as C18:1cis (oleic acid). The saturated fat diet (S) had 9% of energy as palmitic acid, and the trans fatty acid diet (T) had 9% as C18:1trans. Body weight was kept constant throughout the study. After each diet period, insulin pulsatile secretion, insulin sensitivity index (S(I)) by the minimal model method, serum lipids, and fat oxidation by indirect calorimetry were measured. RESULTS: Mean S(I) for the M, S, and T diets was 3.44 +/- 0.26, 3.20 +/- 0.26, and 3.40 +/- 0.26 x 10(-4) min(-1). microU(-1). ml(-1), respectively (NS). S(I) decreased by 24% on the S versus M diet in overweight subjects but was unchanged in lean subjects (NS). Insulin secretion was unaffected by diet, whereas total and HDL cholesterol increased significantly on the S diet. Subjects oxidized the least fat on the M diet (26.0 +/- 1.5 g/day) and the most fat on the T diet (31.4 +/- 1.5 g/day) (P = 0.02). CONCLUSIONS: Dietary fatty acid composition significantly influenced fat oxidation but did not impact insulin sensitivity or secretion in lean individuals. Overweight individuals were more susceptible to developing insulin resistance on high-saturated fat diets.