Effects of gamma-linolenic acid and oleic acid on paclitaxel cytotoxicity in human breast cancer cells.

It has been suggested that dietary interventions may improve the effectiveness of cancer chemotherapy. We have examined the combined in vitro cytotoxicity of paclitaxel and the fatty acids gamma-linolenic acid (GLA, 18:3n-6) and oleic acid (OA, 18:1n-9) in human breast carcinoma MDA-MB-231 cells. The effect of fatty acids on paclitaxel chemosensitivity was determined by comparing IC(50) and IC(70) (50 and 70% inhibitory concentrations, respectively) obtained when the cells were exposed to IC(50) and IC(70) levels of paclitaxel alone and fatty acids were supplemented either before or during the exposure to paclitaxel. The 3-4,5-dimethylthiazol-2-yl-2,5-diphenyl-tetrazolium bromide (MTT) assay was used to determine cell growth inhibition. GLA by itself showed antiproliferative effects, and a possible GLA-paclitaxel interaction at the cellular level was assessed by the isobologram and the combination-index (CI) methods. Isobole analysis at the isoeffect levels of 50 and 70% revealed that drug interaction was predominantly synergistic when GLA and paclitaxel were added concurrently for 24 h to the cell cultures. Interaction assessment using the median-effect principle and the combination-index (CI) method showed that exposure of MDA-MB-231 cells to an equimolar combination of concurrent GLA plus paclitaxel for 24 h resulted in a moderate synergism at all effect levels, consistent with the results of the isobologram analysis. When exposure to GLA (24 h) was followed sequentially by paclitaxel (24 h) only an additive effect was observed. The GLA-mediated increase in paclitaxel chemosensitivity was only partially abolished by Vitamin E, a lipid peroxidation inhibitor, suggesting a limited influence of the oxidative status of GLA in achieving potentiation of paclitaxel toxicity. When OA (a non-peroxidisable fatty acid) was combined with paclitaxel, an enhancement of chemosensitivity was found when OA was used concurrently with paclitaxel, although less markedly than with GLA. Pretreatment of MDA-MB-231 cells with OA for 24 h prior to a 24 h paclitaxel exposure produced greater enhancement of paclitaxel sensitivity at high OA concentrations than the concurrent exposure to OA and paclitaxel. The OA-induced sensitisation to paclitaxel was not due to the cytoxicity of the fatty acid itself. When these observations were extended to three additional breast carcinoma cell lines (SK-Br3, T47D and MCF-7), simultaneous exposure to GLA and paclitaxel also resulted in synergism. GLA preincubation followed by paclitaxel resulted in additivity for all cell lines. Simultaneous exposure to paclitaxel and OA enhanced paclitaxel cytotoxicity in T47D and MCF-7 cells, but not in SK-Br3 cells, whereas preincubation with OA failed to increase paclitaxel effectiveness in all three cell lines. For comparison, the effects of other fatty acids on paclitaxel chemosensitivity were examined: GLA was the most potent at enhancing paclitaxel cytotoxicity, followed by alpha-linolenic acid (ALA; 18:3n.3), eicosapentaenoic acid (EPA; 20:5n-3) and docosahexaenoic acid (DHA; 22:6n-3), whereas linoleic acid (LA; 18:2n-6) did not increase paclitaxel toxicity. These findings provide experimental support for the use of fatty acids as modulators of tumour cell chemosensitivity in paclitaxel-based therapy.

Expression and purification of human stromelysin 1 and 3 from baculovirus-infected insect cells.

Stromelysin 1 (ST1) is a member of the matrix metalloproteinase (MMP) family probably involved in extracellular matrix degradation. Stromelysin 3 (ST3), considered by sequence homology to be a member of the MMP family of proteases, is specifically expressed in the stroma adjacent to the invasive tumoral cells, but its role in cancer progression remains to be elucidated. Genes encoding ST1 and ST3 were expressed in lepidopteran insect cells using the baculovirus expression vector system. Recombinant baculoviruses were obtained after cloning the full-length cDNA of ST1 and ST3 in plasmids pBacPAK1 and pBacPAK9, respectively. Sf9 insect cells infected with the recombinant baculovirus overexpressed the zymogen proST1 (60 kDa) in an insoluble form, a peak of expression being reached from 24 h postinfection. After solubilization in 8 M urea, and further refolding, activation, and purification, 0.3 mg of mature ST1 (30 kDa), purified to 90% homogeneity, was obtained per 5 x 10(8) infected cells. Recombinant ST1 exhibited proteolytic activity on alpha2-macroglobulin, casein, fibronectin, alpha1-antitrypsin, and laminin. The recombinant zymogen proST3 (55 kDa) was expressed as a soluble form in insect cells, maximal expression occurring at 72 h postinfection. After purification to 95% homogeneity, 2.5 mg of proST3 was obtained per 5 x 10(8) infected cells. A number of proteases including plasmin, urokinase, and ST1 were shown to be able to cleave proST3 giving rise to defined bands of 50-30 kDa. The ST3 mature form of 45 kDa (mST3) was also expressed in the baculovirus system and the obtained protein, 2. 5 mg per 5 x 10(8) infected cells purified to 80% homogeneity, was shown to be active on both casein degradation and alpha2-macroglobulin entrapment assays. Our results suggest that the baculovirus system offers a convenient and efficient means to produce ST1 and ST3 in order to carry out further biochemical studies.