Attachment, invasion, chemotaxis, and proteinase expression of B16-BL6 melanoma cells exhibiting a low metastatic phenotype after exposure to dietary restriction of tyrosine and phenylalanine.

We previously reported that low levels of tyrosine (Tyr) and phenylalanine (Phe) alter the metastatic phenotype of B16-BL6 (BL6) murine melanoma and select for tumor cell populations with decreased lung colonizing ability. To more specifically characterize the effects of Tyr and Phe restriction on the malignant phenotype of BL6, we investigated in vitro attachment, invasion, proteinase expression, and chemotaxis of high and low metastatic BL6 variants. High metastatic variant cells were isolated from subcutaneous tumors of mice fed a nutritionally complete diet (ND cells) and low metastatic variant cells were isolated from mice fed a diet restricted in Tyr and Phe (LTP cells). Results indicate that attachment to reconstituted basement membrane (Matrigel) was significantly reduced in LTP cells as compared to ND cells. Attachment to collagen IV, laminin, and fibronectin were similar between the two variants. Invasion through Matrigel and growth factor-reduced Matrigel were significantly decreased in LTP cells as compared to ND cells. Zymography revealed the presence of M(r) 92,000 and M(r) 72,000 progelatinases, tissue plasminogen activator, and urokinase plasminogen activator in the conditioned medium of both variants; however, there were no differences in activity of these secreted proteinases between the two variants. Growth of the variants on growth factor-reduced Matrigel similarly induced expression of the M(r) 92,000 progelatinase. The variants exhibited similar chemotactic responses toward laminin. However, the chemotactic response toward fibronectin by LTP cells was significantly increased. MFR5, a monoclonal antibody which selectively blocks function of the alpha 5 chain of the alpha 5 beta 1 integrin, VLA-5, decreased the chemotactic response toward fibronectin of ND cells by 37%; the chemotactic response by LTP cells was reduced by 49%. This effect was specific for fibronectin-mediated chemotaxis since the chemotaxis toward laminin and invasion through Matrigel were not altered by the presence of MFR5. The surface expression of VLA-5 was significantly increased in LTP cells as compared to ND cells by flow cytometric analysis. These observations suggest that limitation of Tyr and Phe either directly modifies BL6 or selects for subpopulations with altered in vitro invasion, chemotaxis, and integrin expression.

Tyrosine and phenylalanine restriction sensitizes adriamycin-resistant P388 leukemia cells to adriamycin.

Cancer chemotherapy frequently fails, because tumors develop multiple drug resistance (MDR). Pharmacological efforts to reverse this MDR phenotype and sensitize resistant tumor cells have utilized verapamil (VER) to inhibit p-glycoprotein function and buthionine sulfoximine (BSO) to inhibit glutathione synthesis. Our previous results indicate that restriction of two amino acids, tyrosine (Tyr) and phenylalanine (Phe), may potentially suppress the MDR phenotype. These results show that in vivo Tyr and Phe restriction improves the therapeutic response of a metastatic variant of B16-BL6 (BL6) murine melanoma to adriamycin (ADR) and B16 melanoma to levodopa methyl ester. We examine whether in vitro limitation of Tyr and Phe suppresses ADR resistance of BL6 cells and whether Tyr-Phe modulation of the MDR phenotype is applicable to other tumor types, particularly P388 murine leukemia. Mechanisms underlying Tyr-Phe modulation of ADR resistance are examined in the presence of VER and BSO, singly and in combination. Our results indicate that in vitro Tyr and Phe restriction has no effect on BL6 resistance to ADR. However, Tyr and Phe restriction does increase the sensitivity of ADR-resistant P388 cells to ADR without affecting drug efflux, ADR uptake, or glutathione levels. In addition, this enhanced ADR sensitivity of P388 cells is even more pronounced in the presence of BSO. Suppression of ADR resistance in P388-resistant cells by Tyr and Phe restriction indicates that Tyr- and Phe-mediated modulation of the MDR phenotype is possible and that Tyr and Phe restriction may be useful as a potential adjuvant to effective cancer chemotherapy.

Evidence for nutrient modulation of tumor phenotype: impact of tyrosine and phenylalanine restriction.

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.

Differential growth factor production, secretion, and response by high and low metastatic variants of B16BL6 melanoma.

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)

Modulation of B16-BL6 murine melanoma metastatic phenotype by tyrosine and phenylalanine restriction in the absence of host selection pressures.

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.

Alcohol consumption suppresses metastasis of B16-BL6 melanoma in mice.

Female C57BL/6 mice were fed a defined, pelleted diet and given 10% w/v or 20% w/v ethanol in their drinking water. Natural killer (NK) cell cytolytic activity was compared between water-drinking and ethanol-consuming mice and in mice that were also treated with polyinosinic-polycytidylic acid (poly I:C) to augment NK cell activity or with anti-NK1.1 antibody to decrease activity. NK cell cytolytic activity was not altered in mice given 10% ethanol, but was decreased in mice given 20% ethanol compared to water-drinking mice. Poly I:C treatment increased and anti-NK1.1 antibody treatment decreased NK cell activity in both water-drinking and 20% ethanol-consuming mice. Experimental and spontaneous metastases of B16-BL6 melanoma were evaluated as a function of the duration of ethanol consumption before tumor inoculation and as a function of altered NK cell activity. Experimental metastasis was inhibited after 4 and also after 6.5 weeks of ethanol exposure. Poly I:C treatment inhibited tumor lung colonization irrespective of ethanol consumption. Anti-NK1.1 antibody treatment increased metastasis, although to a lesser degree in mice consuming 10% ethanol. Spontaneous metastasis was inhibited in mice consuming 10% ethanol for 4 weeks, and in mice consuming 20% ethanol for 1 and 4 weeks before melanoma inoculation.

Antimetastatic activity of boro-amino acid analog protease inhibitors against B16BL6 melanoma in vivo.

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.

Specificity of the suppression of metastatic phenotype by tyrosine and phenylalanine restriction.

Amino acid restriction modulates tumor growth, although effects on metastasis are poorly documented. We demonstrate that low levels of tyrosine (Tyr) and phenylalanine (Phe) suppress metastasis of B16-BL6 melanoma and that these effects are specific to these two amino acids. Weight loss and sustained low body weight in mice fed low Tyr and Phe diet do not contribute to the antimetastatic effects. Furthermore, methionine (Met) restriction, which decreased survival of mice inoculated i.p. with B16 melanoma, only slightly inhibited spontaneous metastasis compared to the dramatic inhibition during Tyr and Phe restriction. Tyr and Phe restriction inhibited spontaneous metastasis by impairing the ability of tumor cells to establish metastatic foci and not via differential tumor cell removal from the blood. Spontaneous metastasis is blocked by Tyr and Phe intervention even in mice with established lymph node tumors. Tumors isolated from mice fed low Tyr and Phe diet reinoculated into mice fed normal diet exhibited lower experimental metastatic potential, reflected by decreased formation of lung tumor colonies and increased survival of inoculated mice. This decrease in metastatic potential is not associated with tumor chemosensitivity. These findings indicate that Tyr and Phe restriction could become an important adjuvant to effective melanoma treatment.

Phenotypic stability of B16-BL6 melanoma exposed to low levels of tyrosine and phenylalanine.

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.

Contribution of the extracellular matrix to growth properties of cells from a preneoplastic outgrowth: possible role of hyaluronic acid.

Hyaluronic acid (HA) accumulates around actively growing normal and tumorigenic mammary epithelial cells and has been implicated as a modulator of cell proliferation. We have tested the role of exogenous HA presented in several different forms in in vitro growth regulation of a cell line (CL-S1) derived from preneoplastic mouse mammary tissue. This cell line grows slowly and synthesizes very little HA. We first assessed growth of CL-S1 cells seeded onto actual matrix generated by CL-S1 cells themselves (which has a low HA content) or by a related tumorigenic cell line, +SA, that generates an HA-rich matrix. Growth on both these HA-containing substrata was significantly enhanced above control values on plastic. Growth on the +SA biomatrix was over 5 times greater than on tissue culture plastic and significantly greater than that seen with all other treatments. Differences in growth responses of CL-S1 cells seeded atop CL-S1- and +SA-derived matrices could be attributable to differences in matrix HA content. As a more direct test of this possibility, growth responses of CL-S1 cells to HA covalently bonded to tissue culture dishes and to HA dissolved in culture media were tested. Growth on the prepared HA substrata was consistently twice that on plastic. In soluble form, HA at a concentration of 100 micrograms HA/ml culture medium, stimulated CL-S1 growth 196 and 125% of control in monolayer cultures, respectively, seeded at low (approximately equal to 10(2) viable cells/cm2) and high (approximately equal to 10(4) viable cells/cm2) densities on plastic. Higher HA concentrations inhibited growth at low seeding densities.(ABSTRACT TRUNCATED AT 250 WORDS)