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High concentrations of specific catechins [epigallocatechin gallate (EGCG), epigallocatechin (EGC) and epicatechin gallate (ECG)] inhibit the proliferation of many different cancer cell lines. The aim of this work was to determine if low concentrations of catechins with and without 4-hydroxytamoxifen (4-OHT) co-treatment would cause significant cytotoxicity in estrogen receptor-positive (ERalpha+) and -negative (ERalpha-) human breast cancer cells. Therefore, MCF-7, T47D, MDA-MB-231 and HS578T cells were incubated with EGCG, EGC or ECG (5-25 microM) individually and in combination with 4-OHT for 7 days. Cell number was determined by the sulforhodamine B cell proliferation assay. As single agents, none of the catechins were cytotoxic to T47D cells, while only EGCG (20 microM) elicited cytotoxicity in MCF-7 cells. Additionally, no benefit was gained by combination treatment with 4-OHT. ERalpha- human breast cancer cells were more susceptible as all three catechins were significantly cytotoxic to HS578T cells at concentrations of 10 microM. In this cell line, combination with 4-OHT did not increase cytotoxicity. However, the most striking results were produced in MDA-MB-231 cells. In this cell line, EGCG (25 microM) produced a greater cytotoxic effect than 4-OHT (1 microM) and the combination of the two resulted in synergistic cytotoxicity. In conclusion, low concentrations of catechins are cytotoxic to ERalpha- human breast cancer cells, and the combination of EGCG and 4-OHT elicits synergistic cytotoxicity in MDA-MB-231 cells.

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.

Polyunsaturated fatty acids are cytotoxic to ZR-75-1 human breast tumor cells in culture. This effect may be potentiated by the simultaneous addition of iron. When cytotoxicity was measured in the presence of different concentrations of both gamma-linolenic acid and ferrous chloride there was an increase in cell death above concentrations of 9 microM and 0.05 microM, respectively. The potentiation of the effects of 18:3n-6 at low concentrations by the simultaneous addition of Fe(II) ions supports the contention that an alteration in the intracellular Fe(II)/Fe(III) ratio is necessary to promote autocatalytic lipid peroxidation.

We have investigated the nature of lipid peroxidation occurring in association with cancer-killing produced by gamma-linolenic acid (GLA) and iron (Fe) in cultured human breast cancer cells (ZR-75-1: ZR). UV-spectrophotometry, high performance liquid chromatography (HPLC) and gas chromatography (GC) or gas chromatography-mass spectrometry (GC-MS) have been used to analyze lipid peroxides and their derivatives. Formation of conjugated dienes (CD), the conversion of triphenylphosphine (TPP) to its oxide (TPPO), and the simultaneous production of hydroxy polyunsaturated fatty acids (PUFA-OHs) from these corresponding PUFAs hydroperoxides (PUFA-OOHs) were analyzed in the total lipid extract of ZR cells and of normal human skin fibroblasts (CCD-41Sk:Sk). Fe enhanced the formation of both the CD and TPPO and increased the percentage of dead cells, while vitamin E inhibited these effects. Neither of these events was observed at any significant level in Sk cells. Identity of PUFA-OHs was confirmed by determining the regional positions of the hydroxyl group by GC-MS analysis of hydrogenated methyl ester tert-butyldimethylsilyl ether alcohol derivatives (Me-H2-PUFA-O-TBDMS). The regional isomers identified were 15-, 12- and 8-OH 20 carbon and 13-OH 18 carbon fatty acid derivatives. These results suggest that the increased formation of conjugated dienes and/or hydroperoxyl (or peroxyl) groups in PUFA molecules is relevant to the cancer cell-killing effect of GLA+Fe.