Effects of polyunsaturated fatty acids and their n-6 hydroperoxides on growth of five malignant cell lines and the significance of culture media.

We examined effects of polyunsaturated fatty acids (PUFA), their corresponding hydroperoxy fatty acids (hp-PUFA), as well as various pro- and antioxidants on the growth of tumor cells in culture. When cultured in RPMI 1640 medium, A-427 and WEHI clone 13 cells were both highly sensitive to hydroperoxy docosahexaenoic acid (hp-DHA), but they were far less sensitive in minimum essential medium (MEM). In contrast, A-427 cells were also sensitive to DHA in both culture media, while WEHI clone 13 cells, as well as other cell lines, tested in their respective media, were resistant. The lower sensitivity of the cell lines to hp-DHA in MEM-medium was apparently due to a more rapid reduction of hp-DHA to the corresponding hydroxy-DHA in MEM-medium. Addition of glutathione (GSH) to the culture medium abolished the effects of hp-DHA, but not the effects of DHA, while depletion of intracellular GSH levels by L-buthionine-S,R-sulfoximine strongly enhanced the cytotoxic effect of hp-DHA, but not the cytotoxic effect of DHA. alpha-Tocopherol protected A-427 cells against the toxic effect of DHA and abolished the induced lipid peroxidation, while it did not protect against the toxic effects of hp-DHA in A-427 or WEHI clone 13 cells. Ascorbic acid reduced the cytotoxic effect of DHA, but potentiated the toxic effect of hp-DHA while selenite essentially abolished the toxicity of both DHA and hp-DHA. These results indicate that sensitivity of tumor cell lines to PUFA and their oxidation products depends on their antioxidant defense mechanisms, as well as culture conditions, and establishes hp-DHA as a major, but probably not the sole, metabolite responsible for cytotoxicity of DHA.

Evidence that changes in Se-glutathione peroxidase levels affect the sensitivity of human tumour cell lines to n-3 fatty acids.

The human lung adenocarcinoma cell line A-427 is significantly more sensitive to cytotoxic lipid peroxidation products of eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) than the human lung adenocarcinoma cell line SK-LU-1, and the glioblastoma cell lines A-172 and U-87 MG. The cytotoxic effect as well as lipid peroxidation were abolished by vitamin E. The differential sensitivities of the cell lines were not correlated to the levels of lipid peroxidation products (measured as the end product malondialdehyde), indicating differences in sensitivities to products of lipid peroxidation. The high sensitivity of A-427 is apparently due to a low level of selenium-dependent glutathione peroxidase (GSH-Px), because pretreatment with sodium selenite (250 nM) increased the GSH-Px activity 3- to 4-fold and protected the cells almost completely against the growth inhibitory effect of DHA. Furthermore, 2-phenyl-1,2-benzisoselenazol-3(2H)-one (ebselen) a seleno-organic GSH-Px mimic, suppressed the cytotoxic action of DHA to A-427 in a dose dependent manner. Northern analysis demonstrated that pretreatment with sodium selenite (250 nM) was accompanied by an increased level of GSH-Px mRNA (1.8-fold) in A-427 cells, while the level remained unchanged under the same conditions in DHA/EPA-resistant A-172 cells. In addition, the level of selenophosphate synthetase mRNA (SelD), a key intermediate in tRNA(Sec) formation, increased 1.2- to 1.7-fold in A-427 and A-172 cells after pretreatment with sodium selenite. These results indicate that upregulation of GSH-Px activity by sodium selenite in the EPA/DHA sensitive cell line A-427 may be due to an increase in mRNAs for GSH-Px and a precursor important for formation of tRNA(Sec) which is required for incorporation of selenocysteine in GSH-Px during translation. These results demonstrate an important role for GSH-Px in the cellular defence against cytotoxic lipid peroxidation products. Furthermore, measurement of GSH-Px activities in tumour cells may be one useful biochemical predictor for their sensitivities to polyunsaturated fatty acids.

Specificity of hydroperoxy fatty acid inhibition of cell growth and the lack of effect on tumour necrosis factor-induced cytotoxicity in WEHI clone 13 cells.

We have examined whether different omega6-hydroperoxy fatty acids affect tumour cell growth or modulate TNF-induced toxicity in a fatty acid specific way in WEHI clone 13 fibrosarcoma cells. The omega6-hydroperoxides were synthesized from 8 different n - 6 and n - 3 PUFAs by soybean lipoxygenase. The omega6-hydroperoxy fatty acids inhibited cell growth in a concentration-dependent way by a mechanism that is related to the hydroperoxy moiety. Intracellular GSH seemed to protect since the GSH synthase inhibitor L-buthionine-S,R-sulfoximine (BSO) increased cell growth inhibition further. The antioxidants butylated hydroxyanisole (BHA), butylated hydroxytoluene and alpha-tocopherol did not affect the toxicity. The extent of growth inhibition varied between the hydroperoxides, but the difference was relatively small. The most toxic was hydroperoxy-alpha-linolenic acid which reduced cell survival by 56% after 44 h incubation at 35 microM, while the least toxic, hydroperoxy-gamma-linolenic acid, reduced cell survival by only 10%. The data also show that there is no correlation between toxicity and degree of unsaturation of the hydroperoxy fatty acids. None of the 8 different hydroperoxy fatty acids potentiated TNF-induced toxicity. This, together with the differential effects of BHA and BSO on TNF- and hydroperoxy fatty acid toxicity, indicate that neither the hydroperoxides nor their metabolites are involved in mediating or modulating the TNF-effect.