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The present study was undertaken to investigate mechanisms contributing to differences in natural killer (NK) cell activity in moderately endurance-trained and ethanol-consuming mice. Independent of ethanol exposure, NK cell activity in nylon wool-nonadherent (NWNA) splenocytes is lower in trained than in sedentary control mice (Blank et al. J. Appl. Physiol. 72: 8–14, 1992). Reduced activity may result from a generalized loss of cytolytically active cells, redistribution of NK cells from the spleen to other body compartments, or disruption of paracrine regulation of NK cells after removal of nylon wool-adherent cells. To examine these possibilities, NK cell cytolytic activity was determined in nonenriched splenocytes from treadmill-trained and ethanol-consuming mice. Lymphocyte subpopulations in nonenriched splenocytes and NWNA splenocytes were also compared. Peripheral blood lymphocyte subpopulations were determined to examine combined effects of training and ethanol intake on regional distribution of lymphocytes in blood and spleen. NK cell activity in nonenriched splenocytes from trained water-drinking mice was not reduced compared with that in sedentary mice; rather, cytolytic activity was moderately enhanced (17% increase in lytic units, P < 0.05). Training did not change percentages of T-cells, B-cells, and NK [NK1.1+ and large granular lymphocytes (LGL-1+)] cells or the LGL/NK ratio in the spleen and blood. NK cell cytolytic activity was significantly reduced in nonenriched splenocytes from ethanol-consuming mice, independent of training. These findings support the hypothesis that moderate-intensity endurance training influences splenic NK cell function by modulating paracrine regulation of NK cells.(ABSTRACT TRUNCATED AT 250 WORDS)

Previous work demonstrated that splenic natural killer (NK) cell cytolytic activity was suppressed in ethanol-consuming mice within 1 week and for as long as 10 weeks concurrent with ethanol intake. However, it is unknown if suppression of NK cell activity with ethanol consumption results from regional losses in NK cells. The present experiments were designed to examine the effect of ethanol on the percentage and total number of NK cells in the spleen and blood. Data indicate that, after 4 weeks of ethanol intake, NK cell activity of peripheral blood lymphocytes was also suppressed (50% of controls). With short-term (2-week) exposure, the percentage of NK cells in the blood and the spleen remained relatively constant. In the spleen, changes in the number of NK cells reflected generalized fluctuations in lymphocyte numbers rather than variation in the percentage of NK cells. NK cells were most sensitive to the effects of ethanol during long-term (8-week) ethanol intake, as demonstrated by significant reductions in the total percentage of NK 1.1+ cells in the blood, and the LGL-1+ subset in the blood and spleen. These results provide insight into the mechanism of NK modulation by ethanol. Because NK cells were not selectively depleted after 2–4 weeks of ethanol intake, loss of cytolytically active NK cells cannot explain the suppression of in vitro cytolytic activity of cells from ethanol-consuming mice. However, with prolonged ethanol intake (8 weeks), the depletion of NK cells, specifically the LGL-1+ subset, may contribute to the overall suppression of NK cell cytolytic function.

Chronic ethanol consumption can suppress natural killer (NK) cell activity. Exercise after ethanol administration may enhance blood ethanol clearance, which may benefit the immune response. This study examined the effects of moderate intensity endurance training and chronic ethanol consumption (20% wt/vol) on splenic NK cell activity. Mice were assigned to one of four groups: sedentary, water drinking (SED-H2O); sedentary, ethanol consuming (SED-EtOH); trained, water drinking (TR-H2O), and trained, ethanol consuming (TR-EtOH). TR groups ran 60 min/day, 5 days/wk, at 12 m/min for 10 wk. Mice were killed 48 h after exercise. Baseline NK cell activity was suppressed 30% in TR and EtOH groups compared with SED-H2O controls. Activation with recombinant human interleukin-2 increased cytolytic activity in all groups four- to fivefold. These results indicate that training did not abrogate the effects of chronic ethanol consumption on NK cell activity. Furthermore, moderate endurance training may contribute to suppressed nylon wool-enriched NK cell activity in murine splenocytes for as long as 48 h after exercise.

Interferon (IFN) and tumor necrosis factor (TNF) suppress the development of experimental metastasis and when used together, TNF and IFN show synergistic activity. However, the use of TNF is limited by its ability to initiate inappropriate hemostasis. Hemostatic effects are exaggerated by the procoagulant activity of certain tumor cell lines. Therapy with anticoagulants are indicated to block the effects of tumor cell products as well as chemotherapeutic side effects. Heparin is a glycosaminoglycan with diverse biological activity, including the ability to rapidly accelerate the inactivation of active clotting factors. the present studies have explored the therapeutic effects of combining heparin with TNF or interferon on experimental metastasis in mice using a melanoma cell line (B16BL6). Our data indicate that continued heparinization augments the antitumor activity of both interferon and TNF. Alterations of the hemostatic and immune systems play a role in the producing the observed effect.

Nutrients modulate host metabolism that lead to changes in biological and molecular responses. Thus, it is not surprising that tumor cells also are affected by changes in host nutrition. Because tumors have altered or mutated genes, they often respond differently to metabolic changes than the host. Many tumors have altered or specific requirements for amino acids, and restriction often can curtail growth/cell death, invasion, and/or metastasis of tumor cells without detrimental effects on the host. This chapter reviews the effects that various specific amino acid restriction has on tumor cells themselves and the effects on interaction between the host stromal components. The cell signalling pathways that modulate attachment, motility and invasion are discussed. Also included are in vivo studies encompasing the interactions between the host immune response and B16BL6 melanoma during dietary restriction of tyrosine and phenylalanine. Lastly, the potential for amino acid restriction as one element of adjuvant therapy to control cancer progression is discussed.

We previously found that dietary tyrosine (Tyr) and phenylalanine (Phe) limitation significantly decreased the metastatic phenotype of B16BL6 melanoma cells in vivo and decreased the in vitro invasion of these cells. To more specifically characterize the effects of Tyr and Phe deprivation we examined the three steps involved in invasion: attachment to host cells and components, elaboration of proteases that degrade basement membranes, and migration of invading tumor cells. Here we report that B16BL6 melanoma cell invasion through growth factor reduced (GFR) Matrigel is significantly decreased by Tyr and Phe deprivation. Tyr and Phe deprivation in vitro decreased the attachment of B16BL6 melanoma cells to GFR Matrigel, heparin sulfate proteoglycans (HSPG), neonatal murine epidermal (NME) cells and the extracellular matrix (ECM) from these cells. These cells also exhibited a decrease in chemotactic response to fetal bovine serum (FBS). Deprivation of these two amino acids decreased the secretion of urokinase plasminogen activator (uPA) and tissue plasminogen activator (tPA) while plasminogen activator inhibitor (PAI)-1 and -2 were increased in these cells. These observations suggest that Tyr and Phe deprivation decreases the in vitro chemotactic and invasive ability of B16BL6 melanoma cells by decreasing attachment and secreted PA activity and by increasing secreted PAIs in these cells.

Previous studies in our laboratory indicate that alcohol consumption suppresses the metastasis of B16BL6 melanoma, whereas the cytolytic activity of natural killer (NK) cells is decreased in female C57BL/6 mice given 20% w/v alcohol in their drinking water. In the present study, we further evaluated the involvement of NK cells and alcohol consumption in the cytolytic activity of NK cells, the surface expression of NK phenotypic markers, and metastasis of B16BL6 melanoma in C57BL/6 beige (bgJ/bgJ) mutant mice, which possess inherently low NK-cell cytolytic activity.Beige and control (bgJ/+) mice were given either water or 20% w/v of alcohol in drinking water for 6 1/2 to 7 weeks before assay for cytolytic activity, surface marker expression, and inoculation with B16BL6 melanoma intravenously or into the pinna of the ear.NK cytolytic activity was suppressed in beige mice, and alcohol consumption did not modulate further the cytolytic activity. Beige mice had a lower percentage of NK cells in the peripheral blood and spleen than control mice. Peripheral blood lymphocytes from beige mice also exhibited a reduced percentage of CD4+ T lymphocytes. Alcohol consumption similarly reduced the percentages of NK1.1- and LGL-1-expressing lymphocytes in the peripheral blood and spleen and reduced the percentage of CD8+ T lymphocytes in the peripheral blood in both control and beige mice. Tumor lung colonization was increased in beige mice relative to control mice after intravenous inoculation of B16BL6 melanoma. The increase was more pronounced in water-drinking beige mice than in control mice irrespective of alcohol consumption. Tumor lung colonization was significantly decreased (p0.05) by alcohol consumption in one experiment and partially decreased (p = 0.07) in the other. Mice that were inoculated into the pinna of the ear also exhibited a blunted antimetastatic response to alcohol consumption.These data suggest that the presence of the beige mutation diminishes the antimetastatic effect of alcohol consumption and that there is no interaction between alcohol consumption and NK-cell activity in the modulation of lung metastasis of B16BL6 melanoma cells.

This study was designed to examine the relationship between endurance training volume and natural killer cell (NK) cytolytic activity. We hypothesized that a dose dependent relationship exists between forced treadmill training volume and training induced increases in NK cell cytolytic activity.female, Swiss Webster mice were assigned to treadmill control (TC) or treadmill trained groups (n = 10 per group). Trained mice ran at 12 m per min. (8 degrees grade) for: 15 (EX15), 30 (EX30), or 60 minutes (EX60) per day, five days per week for 11 weeks. Splenic NK cell activity was expressed as median lytic unit (LU), median LU per asialo GM1 (AsGM1+) cell, and median LU per spleen.Median NK activity was not significantly increased by training volume. A trend toward greater median LU per AsGM1+ cell was observed in EX30 group versus TC (p = 0.1). Training volumes less than or greater than this level produced smaller increases in NK cytolytic activity.These data provide preliminary evidence indicating that training induced increases in splenic NK cell cytolytic activity do not exhibit a dose dependent relationship with treadmill training volume.

We found previously that restriction of tyrosine (Tyr) and phenylalanine (Phe) inhibited growth and metastasis of B16BL6 murine melanoma and arrested these cells in the G0-G1 phase of the cell cycle. Here, we report that deprivation of these two amino acids in vitro induces apoptosis in B16BL6 and in human A375 melanoma cells but not in nontransformed, neonatal murine epidermal cells or human infant foreskin fibroblasts. Four days after deprivation of Tyr and Phe in vitro, 37% of B16BL6 and 51% of A375 melanoma cells were undergoing apoptosis. Apoptosis was not associated with elevation in intracellular calcium or alteration in p53 or c-myc protein expression. Expression and Tyr phosphorylation of focal adhesion kinase (FAK) were inhibited in both melanoma cell lines by deprivation of Tyr and Phe but not by deprivation of glutamine or serum. Tyr phosphorylation of FAK in Tyr- and Phe-deprived melanoma cells was enhanced within 30 min of refeeding with complete DMEM. FAK protein expression recovered within 60 min, and cell viability recovered within 24 h. Genistein, a tyrosine kinase inhibitor that specifically inhibits Tyr phosphorylation of FAK, did not induce apoptosis in A375 melanoma cells at a concentration of 50 microM. Genistein prevented the recovery of cell viability upon refeeding with Tyr and Phe to previously deprived A375 melanoma cells. These data collectively indicate that apoptosis induced by Tyr and Phe deprivation is FAK-dependent.

The nm23 gene has been described as a potential metastasis suppressor gene in certain rodent and human tumors. We previously demonstrated that tyrosine and phenylalanine restriction suppresses metastatic heterogeneity of B16-BL6 murine melanoma and selects for tumor variants with decreased metastatic potential. In this study, we investigated nm23 expression in the highly metastatic B16-BL6 (ND) melanoma, its nutritionally derived poorly metastatic (LT) variant, and the syngeneic non-tumorigenic Mel-ab melanocytes. No differences in nm23 expression were observed between ND and LT cells, and nm23 expression varied between different isolates. Previously, we showed that metastatic potential of 1-ND cells decreases and is not altered in 1-LT cells after prolonged in vitro cell passage; however, nm23 expression is equivalently increased by 2-fold. In 2-ND and 2-LT cells, expression of nm23 is not different at higher in vitro cell passage. Expression of nm23 decreased about 2-fold when phorbol 12-myristate 13-acetate (PMA) was removed from Mel-ab cells, which induces these cells to become quiescent. Although membrane-associated protein kinase C (PKC) activity decreased after prolonged PMA treatment in all cells, neither nm23 expression nor proliferation of ND and LT cells was affected by PMA. These data indicate that nm23 expression is related to proliferative activity rather than to the suppression of metastatic potential.

Ethanol (20% w/v) given to female C57BL/6 mice in their drinking water reduces splenic natural killer (NK) cell cytolytic activity after 2, 4, and 10 weeks of consumption. This reduction is transient because the levels of NK cell cytotoxicity from ethanol-consuming mice are nearly equal to those of water-drinking mice after splenocytes were incubated in 1000 IU/ml of recombinant interleukin-2 (rIL2) for 16-18 hr. In this study, mice were given 20% w/v ethanol in the drinking water for 2 weeks. Splenic NK cells were enriched up to 88% by negative selection based on surface expression of NK1.1. Enriched NK cells were expanded in rIL2 for 6 days. Lymphokine-activated killer (LAK) cells from both ethanol-consuming and water-drinking mice were > 95% NK1.1+. LAK cell cytolytic activity was significantly lower against NK-insensitive P815 mastocytoma [6.67 +/- 2.18 vs. 17.21 +/- 1.8 lytic units (LUs), p < 0.01], moderately NK-sensitive B16 melanoma (25.3 +/- 6.6 vs. 66.2 +/- 14.2 LU, p < 0.05), and NK-sensitive YAC-1 lymphoma targets (80.5 +/- 34.7 vs. 177.0 +/- 43.6 LU, p < 0.005) in cells from ethanol-consuming mice compared with water-drinking controls. Ethanol consumption did not affect the morphology or phenotype of LAK cells with respect to surface expression of NK1.1, B220, CD3, CD25, CD11a, CD54, CD45RB, or class I major histocompatibility complex.

Ethanol (20% w/v) given to female, C57BL/6 mice in their drinking water was previously shown to suppress natural killer (NK) cell cytolytic activity in a mixed splenocyte population. The present study was designed to examine the hypothesis that ethanol consumption independently results in inhibition of NK cell cytolytic activity. Mice were given 20% w/v ethanol in the drinking water for 2 weeks, and splenic NK cells were enriched up to 88% based on surface expression of NK1.1. Cytolytic activity of these freshly enriched NK cells from ethanol-consuming mice against YAC-1 lymphoma cells was inhibited an average of 41% relative to water-drinking controls. Cytolytic activity of enriched NK cells from ethanol-consuming mice was stimulated to levels equal to control water-drinking mice after 16- to 18-hr incubation in 1000 units/ml recombinant interleukin 2. These data indicate that in vitro cytolytic activity of NK cells from ethanol-consuming mice is inhibited in the absence of other modulatory leukocytes.

We have shown that Tyr and Phe restriction suppresses the malignant phenotype of the highly invasive and metastatic BL6 variant of B16 murine melanoma. Lung-colonizing abilities of Tyr- and Phe-modulated in vivo and in vitro variants of BL6 are inhibited following intravenous inoculation into mice fed normal diet. Although this antimetastatic effect of Tyr and Phe restriction is most likely not due to differences in attachment to endothelium, our data indicate that major impacts of Tyr and Phe restriction are at the level of the tumor, itself. Modulation of host immune responses, which in turn suppresses metastasis, does not appear to contribute significantly to the altered phenotype. Although numbers and function of T cells, mast cells, and NK cells are affected by Tyr and Phe restriction, they are not involved in the Tyr- and Phe-mediated suppression of tumor growth, metastasis, or angiogenesis. Our data do not rule out the importance of other host factors involved in the Tyr and Phe modulation of tumor phenotype. The outcome of this modulation results most likely from complex Tyr/Phe-tumor-host interactions.

Low levels of tyrosine and phenylalanine alter the metastatic phenotype of B16BL6 murine melanoma. In this study, we investigated expression and secretion of fibroblast growth factor-like (FGF-like) and transforming growth factor beta-like (TGF beta-like) molecules as well as the biological effect of basic FGF (bFGF) and TGF beta 1 on high (NDP) and low (LTP) metastatic variants of B16BL6 melanoma. Both NDP and LTP cells expressed bFGF-like and TGF beta-like polypeptides as detected by Western blot analysis. An M(r) 29,000 bFGF-like form eluted from heparin-Sepharose by 0.6 M NaCl was found in extracts of both NDP and LTP cells. Elution at 0.6 M NaCl suggested that this M(r) 29,000 form might be more closely related to FGF-5 than to bFGF. In addition, cell extracts of LTP, but not NDP cells, contained an M(r) 47,000 monomeric bFGF-like form that was not retained on heparin-Sepharose. Three major specific immunoreactive forms of M(r) 44,000, 36,000, and 29,000 were present in conditioned medium from NDP cells. The M(r) 29,000 form present in the conditioned medium of NDP cells was retained on heparin-Sepharose. Only the M(r) 44,000 and 36,000 FGF-like molecules were detected in conditioned medium from LTP cells, and they were also not retained on heparin-Sepharose. Anti-TGF beta antibody that recognized both TGF beta 1 and TGF beta 2 detected 3 different TGF beta-like forms (M(r) 25,000, 23,000 and 22,000) in NDP and LTP cell extracts. Conditioned medium from NDP cells contained an M(r) 38,000 form of TGF beta; however, no immunoreactive forms were found in conditioned medium from LTP cells. Thus, the NDP-LTP differences in this melanoma system were primarily in growth factor secretion, not expression. The effect of exogenous bFGF and TGF beta 1 on proliferation of LTP and NDP cells was determined by [methyl-3H]thymidine uptake. bFGF stimulated proliferation of NDP cells; whereas, LTP cells exhibited no increase in proliferation. Both NDP and LTP cells responded to TGF beta 1. Proliferation of NDP cells was inhibited more by this growth factor than was proliferation of LTP cells. When NDP and LTP cells were incubated with 5 ng/ml TGF beta 1 and various amounts of bFGF, the effect of TGF beta 1 was masked. Antibody depletion of bFGF-like molecules from NDP conditioned medium resulted in the decreased proliferation of NDP cells but not LTP cells. Depletion of TGF beta-like molecules resulted in increased proliferation of LTP cells but did not affect NDP cells.(ABSTRACT TRUNCATED AT 400 WORDS)

We previously showed that restriction of tyrosine (Tyr) and phenylalanine (Phe) in vivo dramatically suppresses the metastatic phenotype of B16-BL6 (BL6) murine melanoma. Present results indicate a direct effect of Tyr and Phe restriction on the tumor in the absence of host selection pressures. Lung colonizing ability of BL6 is dramatically suppressed after one passage in vitro in media containing low levels of Tyr and Phe. This antimetastatic effect is immediate, stable for at least 5 in vitro passages in Tyr and Phe restricted media, and evident event after levels of Tyr and Phe are restored to normal. Heterogeneity for lung colonizing ability is suppressed, as evidence by fewer tumor colonies formed by clones following i.v. inoculation into mice fed normal diet. This suppression of BL6 metastatic phenotype is not due to differential clearance and retention in the lung or to decreased growth, but is specific for these two amino acids. As the mechanism(s) for the antitumor effects of Tyr and Phe restriction are detailed, the relevance of Tyr and Phe restriction as an early adjuvant to effective cancer treatment can be explored.

A crucial requirement for effective boron neutron capture therapy (BNCT) of cancer is the selective localization of high concentrations of boron in tumor tissue relative to adjacent normal tissue, thus minimizing damage to normal tissues caused by the neutron beam. Several promising boron compounds are presently available as boron-delivery agents for neutron capture therapy of malignant melanoma. More are expected to be developed in the near future. Therefore, there is a need to experimentally describe these boron-delivery compounds in vitro and in vivo, to screen these agents by critically comparing their efficacies with known boronated agents, and to effectively optimize their BNCT potential.

Liposomes, of a specific size and composition, have demonstrated unique ability to selectively deliver significant quantities of boron to murine tumors. Aqueous solutions of water soluble compounds with a high boron content have been encapsulated into the liposomes and studied in vivo. Although the liposomes deliver their contents to the interior of the tumor cells, the characteristics of the boron compound determine whether boron will be retained by the tumor cells or simply cleared from the system. Investigation of a series of compounds which vary in characteristics such as size, charge, and reactivity have allowed us to develop a working hypothesis which predicts a priori, with some degree of success, the ability of these boron compounds to be retained by tumor cells.

Di- and tripeptide boro-amino acid analog protease inhibitors with specificity for chymotrypsin and elastase decrease the number of melanotic foci formed in the lungs of mice in the B16BL6 experimental metastatic tumor model. These effects were at significantly lower concentration than leupeptin or other natural chymotrypsin inhibitors previously reported. These results support the involvement of elastase and chymotrypsin in the metastatic process.

Cell-associated boron levels adequate for neutron capture therapy (NCT) have been demonstrated in-vitro using cultured melanoma cells(1) and in-vivo using xenografts in mice.(2-5) Preliminary in-vivo studies performed by researchers at the College of Veterinary Medicine, Washington State University (WSU), using a spontaneous canine melanoma model, showed subtherapeutic tumor concentrations of para-boronophenylananine (p-BPA) in a large proportion of dogs. Possible explanations include poor solubility of p-BPA at physiological pH, physiological differences between transplanted and spontaneous tumors, and lack of metabolic incorporation at the cellular level.(2-5)

We previously demonstrated that tyrosine (Tyr) and phenylalanine (Phe) restriction suppresses metastatic heterogeneity of B16-BL6 (BL6) melanoma and selects for tumor variants with decreased metastatic potential. In this study, we investigate stability of this Tyr- and Phe-modulated tumor phenotype by sequentially transplanting BL6 in vivo into mice fed Low Tyr and Phe Diet. Metastatic potential of BL6 is suppressed after one subcutaneous passage. Suppression is unlikely to result from inhibition of tumor growth, since growth in vitro is significantly increased. The metastatic potential of the Tyr- and Phe-modulated tumor is unstable after in vivo passage, and lung colonizing ability is regenerated after ten in vivo passages. Conversely, the antimetastatic effect of Tyr and Phe restriction is stable after prolonged in vitro passage. The metastatic potential of tumors from mice fed Normal Diet is unstable after long-term in vitro culture. Sensitivity to adriamycin of BL6 from mice fed Low Tyr and Phe Diet is increased and is not altered by change in metastatic potential.

Holotyrosine phenol-lyase (EC 4.1.99.2), a pyridoxal-5'-phosphate (PLP)- requiring enzyme, was shown to rapidly dissociate when injected into BDF1 mice. The holoenzyme dissociated when incubated in plasma but not 0.01 M potassium phosphate (pH 7.4) buffer at 37 degrees C. A nonspecific alkaline phosphatase from calf intestine was found to inactivate the holoenzyme at pH 7.4 and 37 degrees C. This inactivation was inhibited in the presence of 0.5 M potassium phosphate buffer. Two other PLP-requiring enzymes, aspartate aminotransferase (EC 2.6.1.1), and alanine aminotransferase (EC 2.6.1.2) were inactivated by alkaline phosphatase in a similar manner. Incubation of holotyrosine phenol-lyase in the presence of bovine serum albumin also resulted in a reduction of holoenzyme activity but partially protected the enzyme from inactivation by alkaline phosphatase. A nuclear fraction having PLP-hydrolyzing activity also inactivated holotyrosine phenol-lyase. A regulatory function for alkaline phosphatase in the metabolism of PLP-requiring enzymes is suggested by these data.

Administration of L-tyrosine phenol-lyase inhibited growth of established B16 melanomas in BDF1 mice. Under the conditions used, the enzyme exerted a cytostatic effect on the tumors. The addition of L-tyrosine phenol-lyase to growing melanoma cultures blocked cell division and caused an accumulation of cells in the G1 (G0) pultures was suppressed, the most pronounced effect of L-tyrosine phenol-lyase was a rapid and near-complete cessation of uridine uptake and incorporation. This block in RNA synthesis was also observed in human fibroblasts treated with L-tyrosine phenol-lyase.

The abilities of rodent tumor cell lines; B16BL6, ND and LT dietary variants of B16BL6, +SA, RT7-4bs and RT7-4bLs to invade composite collagen I gels containing heparin, chemically modified heparins, heparan sulfate, chondroitin sulfate, hyaluronic acid, dextran, dextran sulfate, laminin and collagen IV were investigated, and compared to the invasion of plain collagen I gels. The presence of heparin or heparan sulfate most generally promoted tumor cell invasion of the gels, with more aggressive invasion being noted for the more metastatic variants examined. Of the chemically modified heparins tested, carboxyl-reduced heparin promoted matrix invasion by B16BL6 and +SA cells to the greatest degree. Hyaluronic acid marginally promoted invasion by +SA and RT7-4bs primary cells while, in these collagen I based gels laminin only promoted matrix invasion by primary +SA cells to a very limited degree. The tumor cell lines attached relatively poorly to heparan sulfate substrates compared to the other glycosaminoglycans tested, and the primary tumor cell lines also attached relatively poorly to collagen I. As expected, highly metastatic variants showed greater attachment to laminin than did their less metastatic counterparts. Apart from the negative correlation of cellular attachment to heparan sulfate substrates with invasiveness towards heparan sulfate containing gels, no other relationships emerged linking attachment rates with invasive activities for particular complex gel compositions. Our results suggest an important role for heparan sulfate, and possibly also tissue heparin, in promoting tumor cell invasion of extracellular matrices. Results from complex gels containing dextran or dextran sulfate failed to support the hypothesis that GAG sulfation is important to cellular invasion. The activity of the chemically modified heparins in promoting invasion, when present as components of these model matrices, suggests that part of the anti-metastatic activity of these compounds, when preincubated with tumor cells prior to intravenous inoculation, could result from interference with tumor cell extravasation.

An In Vivo model is presented to determine the effects of alpha methyl-para-tyrosine methyl ester (MPT), fusaric acid (FA), and haloperidol on nomifensine-induced seizures. All three drugs were active with haloperidol having the greatest effect on delay of onset to convulsion. The results suggest that both dopamine and norepinephrine are involved in nomifensive-induced convulsions and that administering agents known to lower both dopamine and/or norepinepherine in the rat brain delays onset but that haloperidol, which specifically blocks post-synaptic dopamine receptors, had the greatest activity.

Treatment with the drug combination of levodopa methylester and benserazide, supplemental ascorbate, and dietary deficiencies of tyrosine and phenylalanine more than doubled the median survival time of female (C57BL/6 X DBA/2)F1 mice bearing B16 melanoma tumors. The mechanism for this antitumor effect was not well defined. This study was designed to test the hypothesis that the antitumor activity of levodopa methylester and ascorbate against B16 melanoma is related to the generation of free radicals of oxygen, which peroxidize lipid constituents of cell membranes leading to cell death. As an indication of lipid peroxidation, the individual and combined effects of drug treatment and ascorbate supplementation on host and tumor malondialdehyde levels were examined in mice fed one of three test diets (commercial, purified, or deficient) containing decreasing amounts of tyrosine and phenylalanine. Malondialdehyde levels were increased in the livers of all untreated tumor-bearing mice, which suggests that the tumor alters host antioxidant defenses. Drug treatment and ascorbate supplementation alone and in combination increased hepatic malondialdehyde levels inversely to the amounts of tyrosine and phenylalanine in the diet, and the effects of drug and ascorbate on malondialdehyde levels were additive. Plasma levels remained unchanged by drug treatment, ascorbate supplementation, or tumors in mice fed the commercial or purified diets. Higher levels were observed only in tumor-bearing mice fed the deficient diet and given both drug treatment and ascorbate supplementation. Changes in tumor malondialdehyde levels generally correlated with the effects of the drug and ascorbate on survival time of mice bearing B16 melanoma. Tumors from mice fed the commercial diet accumulated little malondialdehyde, and therapy was relatively ineffective in this dietary group. In mice fed purified or deficient diets, drug treatment and ascorbate supplementation alone increased survival and tumor malondialdehyde levels, but the level of peroxidation in mice receiving the ascorbate supplementation was low compared to its greater antitumor effect on B16 melanoma. Although ascorbate enhanced the peroxidative activity of the drug on B16 melanoma tumors, the effects of the drug and ascorbate on malondialdehyde levels were not additive. Ascorbate enhanced survival of tumor-bearing mice that were fed the deficient diet and that were treated with drug, which indicated that ascorbate supplementation acted via other mechanisms.(ABSTRACT TRUNCATED AT 400 WORDS)

The effects of a combined dietary restriction of tyrosine and phenylalanine on metastasis were investigated with the use of 3 rodent tumor cell lines: B16-bladder 6 (BL6) melanoma inoculated into (C57BL/6 X DBA/2)F1 mice, Lewis lung (3LL) carcinoma inoculated into C57BL/6 mice, and RT7-4bs hepatocarcinoma inoculated into BD-IV rats. When examined for effects on spontaneous metastasis, dietary restriction of tyrosine and phenylalanine had no effect on metastasis to draining lymph nodes in either BL6 or 3LL tumors. However, the restriction did reduce metastasis of RT7-4bs cells to draining lymph nodes by 60%. In all tumor systems, the dietary restriction effectively inhibited the subsequent growth of lymph node tumors. The most marked effect of the dietary restriction was on spontaneous hematogenous metastasis, which was almost totally blocked for BL6 cells and was reduced by 85% for RT7-4bs cells. Tumor-associated splenomegaly also was completely inhibited in 3LL tumor-bearing mice. The selective dietary amino acid restriction failed to reduce initial lung colonization in the experimental metastasis assay but clearly inhibited subsequent tumor outgrowth in the lungs. These findings demonstrate that modification of host nutritional status by restriction of the dietary intake of tyrosine and phenylalanine exerts a dramatic antimetastatic effect directed particularly on spontaneous hematogenous metastasis. Although the preliminary data suggest a primary modulating effect on tumor cell populations growing in diet-restricted animals to reduce inherent metastatic ability, the actual mechanisms remain to be defined.

Tyrosine phenol-lyase from Erwinia herbicola was purified with the goal of assessing its effect on growth of malignant melanoma. Ammonium sulfate-sodium citrate fractionation and diethylaminoethyl cellulose-hydroxylapatite chromatography were used. The purified enzyme was shown to reduce plasma tyrosine levels when administered to normal C57BL x DBA/2 F1 mice. The plasma half-life value of the enzyme was found to be 6 to 7 hr. Unlike results reported with glutaminase and asparaginase preparations, the lactate dehydrogenase-elevating virus had no significant influence on plasma clearance of tyrosine phenol-lyase. The enzyme significantly inhibited growth of established B-16 melanoma tumors.

The toxicity of chronic alcohol intake on natural killer (NK) cell activity of spleen cells from well-nourished, female C57BL/6 mice was studied in a 4-hour cytolytic chromium-release assay against YAC-1 lymphoma cells. Mice were fed a nutritionally complete crystalline amino acid diet and received 20% w/v alcohol solution for 12 weeks. Ad libitum and pair-fed control mice were given diet and either an isocaloric glucose solution or water. Decreased NK cell activity was observed in alcohol-consuming mice relative to all other control groups. NK cell activity was moderately decreased by feeding mice a high glucose diet, but more severely lowered in pair-fed groups compared to ad libitum control groups.

Tyrosine phenol-lyase from Erwinia herbicola was purified from a cell-free extract in a single step on Cibacron Blue F3GA-agarose. The protein was purified as the apoenzyme and was unstable after affinity chromatography. Alanine aminotransferase and aspartate aminotransferase from porcine heart also bound to Cibacron Blue F3GA-agarose. These enzymes were partially purified as holoenzymes from a crude porcine heart extract by elution with NADH and KCl. Alanine aminotransferase was purified 19 fold by this procedure.

Procarbazine is a 1,2-disubstituted hydrazine derivative that is used to treat human leukemias. The anticancer activity of procarbazine results from bioactivation to reactive intermediates. It is first oxidized to azoprocarbazine and further N-oxidized to a mixture of methylazoxyprocarbazine and benzylazoxyprocarbazine isomers. In this study the azoxyprocarbazine isomers were synthesized and purified. The cytotoxic effect of the metabolites on the L1210 murine leukemia cell line were then evaluated in vitro by use of a colorimetric assay using 3-(4,5-dimethyl-thiazol-2-yl)-2,5-diphenyl-tetrazolium bromide. The results of this study showed that the methylazoxyprocarbazine isomer was the most cytotoxic metabolite (IC50, 0.2 mM). The benzylazoxy isomer had an insignificant cytotoxic effect, and a mixture of the two isomers was intermediate in effectiveness. This assay, however, could not be used to determine the cytotoxicity of procarbazine since the drug itself (not the live cells) reduced the dye. A soft-agar clonogenic assay demonstrated that procarbazine was cytotoxic only at higher concentrations (IC50, 1.5 mM) than methylazoxyprocarbazine (IC50, 0.15 mM). The effect of procarbazine and its metabolites on the survival of L1210 tumor-bearing mice was determined, and methylazoxyprocarbazine was again the most effective compound. These studies demonstrate that the methylazoxyprocarbazine metabolite is probably the major cytotoxic intermediate involved in the mechanism of anticancer action of procarbazine.

Natural killer (NK) cell activity of spleen cells from female C57BL/6 mice receiving 10% w/v alcohol solution for 4 weeks was studied in mice fed a nutritionally complete crystalline amino-acid diet and in mice fed diets moderately deficient in (i) tyrosine and phenylalanine or (ii) methionine. Natural killer cell activity was determined in a 4-hr cytolytic chromium-release assay against YAC-1 lymphoma cells. Alcohol consumption did not effect NK cell-mediated lysis irrespective of nutritional status; however, NK-cell activity was depressed in mice fed the tyrosine- and phenylalanine-deficient diet and was enhanced in mice fed the methionine-deficient diet. These data suggest that the changes in immune function often observed in alcoholics may be more closely linked to dietary and nutritional status than to the direct effects of the ingested alcohol.

Procarbazine causes dose-dependent decreases in sperm count after a single i.p. injection in (C57BL/6 X DBA/2)F1 male mice. Two antioxidants, N-acetylcysteine and sodium ascorbate, administered with equimolar doses of procarbazine decreased the spermatotoxicity of procarbazine. At the highest doses of procarbazine (400 mg/kg) that caused a 56% decrease in sperm count, equimolar doses of N-acetylcysteine coadministered with procarbazine caused only a 17% decrease in sperm count, and equimolar doses of ascorbate coadministered with procarbazine caused only a 13% decrease in sperm count. Thus, protection against the spermatotoxic effects of procarbazine was demonstrated with either antioxidant. The effect of the antioxidants on the chemotherapeutic efficacy of procarbazine against murine L1210 leukemia was also assessed. Procarbazine at the highest dose (600 mg/kg) increased mean survival time of mice inoculated i.p. with 1 X 10(5) L1210 leukemia cells by 31%. Simultaneous administration of equimolar doses of either N-acetylcysteine or ascorbate given with procarbazine caused no change in the increased mean survival time of tumor-bearing mice. These results indicate a decrease in the toxicity of procarbazine when coadministered with antioxidants, via decreased spermatotoxicity without changing anticancer efficacy. The results also indicate that different mechanisms are involved in the spermatotoxicity and anticancer activity of procarbazine.

A limiting factor in the depletion of plasma tyrosine following tyrosine phenol-lyase injection into normal mice was found to be the availability of an essential cofactor, pyridoxal phosphate. Because of the extremely short half-life of this cofactor, adequate elevation of circulating cofactor levels for prolonged periods by injection of a pyridoxal phosphate solution was not practical. Similarly, long-term diets enriched with pyridoxine and pyridoxal phosphate did not significantly improve the efficiency of the injected holoenzyme. A repository dosage form was devised that consisted of an s.c. implant of pyridoxal phosphate suspended in a spermaceti and peanut oil mixture. Under these conditions a sustained increase in holoenzyme activity levels and a significant resulting decrease in plasma tyrosine levels were obtained.

We report here the single and combined antitumor activity on B16 melanoma in female C57BL/6 X DBA/2F1 mice bearing s.c. tumors of sodium ascorbate, carbidopa-levodopa methyl ester, and dietary phenylalanine and tyrosine deficiency. Groups of 15 mice were fed continuously one of three test diets both with and without sodium ascorbate (30 mg/ml) in the drinking water beginning 2 weeks before inoculation of 10(6) melanoma cells. The test diets included the following amounts of tyrosine-phenylalanine: commercial, 1.09 and 0.64%; purified, 0.6 and 0.3%; and deficient, 0.08 and 0.04%. Drug-treated groups received daily injections of carbidopa (100 mg/kg) and levodopa methyl ester (1000 mg/kg) i.p. for 15 days beginning 1 day after tumor transplant. Tumor growth curves and median survival time were determined. Ascorbate stimulated tumor growth in the commercial diet group. In mice fed the purified diet, ascorbate inhibited growth in some tumors, while it had no effect on others. Ascorbate inhibited tumor growth in mice fed the deficient diet, which itself severely inhibited tumor growth, and in this group increased survival by 82%. Drug treatment had little effect on tumor growth and survival of mice fed the commercial diet, but it significantly decreased growth and moderately increased survival of mice fed the purified diet. The deficient diet enhanced drug activity and increased survival of tumor-bearing mice by 73%. Combined therapy had little effect in mice fed the commercial diet;l however, mice fed the purified diet and receiving drug and ascorbate had smaller tumors and lived 55% longer. In mice fed the deficient diet, the combination retarded tumor growth and increased survival dramatically by 123%. These data indicate that adding ascorbate and restricting tyrosine and phenylalanine in combination with levodopa methyl ester therapy may become an important strategy for treating malignant melanoma.