Mechanisms of the selective cytotoxic actions of certain essential fatty acids.

Polyunsaturated fatty acids (PUFA) have a selective cytotoxic/cytostatic effect on a number of tumor cell lines in culture. Although this process may be enhanced by the addition of iron there is a minimum level of PUFA necessary for potentiation of cell death. Vitamin E blocks PUFA cytotoxicity when added up to 5 days after fatty acid administration. Levels of thio-barbiturate reactive material (TBARM) in the medium rise in parallel with cell death. However, they are not affected by small alterations in temperature or oxygen tension. Incubating cells with PUFA causes marked alterations in the fatty acid patterns of both neutral and phospholipid fractions. Membrane fluidity is increased and the activity of membrane-bound receptors may be influenced directly or through the actions of eicosanoids derived from the exogenous fatty acid. PUFA may be an effective way of influencing tumor growth and a safe approach for the management of human cancer.

Exogenous gamma-linolenic acid alters hormone stimulated cyclic AMP levels in U937 cells.

Polyunsaturated fatty acids are selectively cytotoxic in culture. Incorporation of these fatty acids leads to profound changes in membrane fatty acid composition which in turn may alter the activity of transmembrane receptor/effector systems. In U937 cells, hormone stimulated production of cyclic AMP can be reduced by 30% following incubation with gamma-linolenic acid (18:3n-6). It is suggested that beta-adrenoreceptor number, subtype and adenylyl cyclase stimulation may be regulated by alterations in membrane fatty acid composition as a result of changes in the levels of polyunsaturated fatty acids and alterations in eicosanoid production.

Vitamin E blocks the cytotoxic effect of gamma-linolenic acid when administered as late as the time of onset of cell death--insight into the mechanism of fatty acid induced cytotoxicity.

Certain polyunsaturated fatty acids can selectively kill tumor cell lines while causing little to no harm to normal cell lines. However, the mechanism of this cytotoxicity is only partially understood. Antioxidants such as vitamin E have been shown to be capable of completely blocking the cytotoxic response when administered concomitantly with the fatty acid. We report here that when vitamin E was added as late as 6 days following fatty acid treatment, at a time point when the process of cell death was well underway, any further development of cell death was blocked. This implies that the mechanism of fatty acid induced cytotoxicity does not involve a gradual compromising of the cell over the 5-7 day time course of cell death. Instead, the event triggering cell death is an oxidative phenomenon occurring over a short time span of minutes or hours, not days, and is completely blocked by vitamin E.

Metabolism of n-6 fatty acids by NIH-3T3 cells transfected with the ras oncogene.

N-6 fatty acid metabolism was compared in NIH-3T3 cells and DT cells, which differ only in the presence of the v-Ki-ras oncogene. Non-dividing cells were incubated with [1-14C]-labelled fatty acids (18:2n-6, 18:3n-6, 20:3n-6 and 20:4n-6) at different time intervals (2-24 h) and concentration (0-120 microM). In both cells lines, the uptake of different fatty acids from the medium was similar and reached a maximum at 6-8 h. All fatty acids reached the same maximum level in DT cells, whereas, the relative uptake of added fatty acids by NIH-3T3 cells was different: 20:4n-6 > 20:3n-6 > 18:2n-6 = 18:3n-6. Throughout the incubation (2-24 h), desaturation and elongation of n-6 fatty acids was more active in DT cells than in NIH-3T3 cells. However, in both cell lines, incubated with different n-6 fatty acid precursors, the levels of radiolabelled 20:4n-6 were relatively constant. In DT cells, phosphatidylcholine was found to be the major fraction labelled with n-6 fatty acids precursors and those of endogenous synthesis, whereas, in NIH-3T3 cells the neutral lipid fraction, particularly triglycerides, was also strongly labelled. In concentration dependent studies, phospholipid labelling by fatty acids was saturable. At lower concentrations, especially in DT cells, phospholipids were labelled predominantly. As the concentration increased there was an overflow into the triglyceride fraction. Since the differences in fatty acid metabolism between the two cell lines cannot be related to the growth rate, it is suggested that they were a consequence of the expression of the v-Ki-ras oncogene.

Variations in temperature and oxygen content do not alter levels of thiobarbiturate reactive material in human breast tumour cells (ZR-75-1) incubated with gamma-linolenic acid.

The mechanism by which tumour cells may be killed in vitro by exogenous polyunsaturated fatty acids may involve lipid peroxidation. Gamma-linolenic acid caused a dose and time-dependent reduction in ZR-75-1 cell growth. However, altering either the incubator temperature (35, 37 and 39 degrees C) or the oxygen content (16, 21 and 26%) had little effect on either the growth of cells in the presence of gamma-linolenic acid or on thiobarbiturate reactive material levels over a 7 day period. Thus, small changes in cell culture conditions do not affect 18:3n-6 cytotoxicity or markers of lipid peroxidation.

Metabolism of radiolabelled 18:2n-6 and 18:3n-6 by NIH-3T3 cells and the DT subclone.

The incorporation and metabolism of delta-6-desaturase substrate and product, [1-14C]-linoleic (18:2n-6) and [1-14C]-gamma-linolenic acid (18:3n-6), was examined in NIH-3T3 cells and the DT subclone which differs only in the presence of the v-Ki-ras oncogene. Similar amounts of post delta-6 and delta-5 desaturase metabolites were found in both cell lines indicating that the activity of these important enzymes of fatty acid metabolism was not affected by the expression of the oncogene. However, measurable quantities of the direct elongation product of 18:2n-6, 20:2n-6, were only found in DT cells. Radiolabel was recovered predominantly from the phospholipid fraction at low fatty acid concentrations, whereas neutral lipid labelling occurred when higher concentrations of exogenous fatty acid were present. This effect was most pronounced in DT cells and may result from the presence of the activated ras oncogene.

Comparison of the metabolism of alpha-linolenic acid and its delta 6 desaturation product, stearidonic acid, in cultured NIH-3T3 cells.

The incorporation and metabolism of alpha-linolenic acid (18:3n-3) and its delta 6 desaturase product, stearidonic acid (18:4n-3), were compared by NIH-3T3 cells. In the presence of fetal calf serum, cells accumulated exogenously added 18:3n-3 and 18:4n-3 apparently at the expense of oleic acid (18:1n-9). Both 18:3n-3 and 18:4n-3 were elongated and desaturated to eicosatetraenoic acid (20:4n-3), eicosapentaenoic acid (20:5n-3) and docosapentaenoic acid (22:5n-3), but not to docosahexaenoic acid (22:6n-3), and were incorporated into phospholipids and triacylglycerols. Over a 4-d period, the growth of NIH-3T3 cells was slightly stimulated in the presence of 18:3n-3 (20 micrograms/mL) but was strongly inhibited in the presence of 18:4n-3 at the same concentration. This inhibition may be caused by enhanced lipid peroxidation as a result of the high levels of 18:4n-3 present.

The effect of n-6 fatty acids on normal and v-Ki ras transformed NIH-3T3 cells.

The effect of exogenous gamma-linolenic acid (18:3n-6) was examined on NIH-3T3 and a subclone expressing the v-Ki-ras oncogene (DT). 18:3n-6 inhibited DT cell growth more readily than NIH-3T3 cell growth. In comparison, linoleic acid (18:2n-6) had no effect on the growth of either cell line. DT cells elongated and desaturated both 18:2n-6 and 18:3n-6 to dihomo-gamma-linolenic acid (20:3n-6) and arachidonic acid (20:4n-6) to a much greater extent than NIH-3T3 cells and had a much higher membrane fluidity. The presence of the ras gene or its product appears to increase the metabolism of polyunsaturated fatty acids and potentiate the cytostatic actions of 18:3n-6.