Jeanne M. Connolly

Omega-3 fatty acids as cancer chemopreventive agents

There is both epidemiologic and experimental evidence that the long-chain omega-3 fatty acids (FAs), which occur at high levels in some fish oils, exert protective effects against some common cancers, notably those of breast, colon, and, perhaps, prostate. Multiple mechanisms are involved in this chemopreventive activity, including suppression of neoplastic transformation, cell growth inhibition and enhanced apoptosis, and antiangiogenicity; however, a common feature of most of these biological effects is the inhibition of eicosanoid production from omega-6 FA precursors. Several of the known risk factors for breast, and colon cancer may be favorably modified by dietary omega-3 FA supplementation, and the implementation of clinical chemoprevention trials is now feasible.

Regulation of tumor angiogenesis by dietary fatty acids and eicosanoids.

Angiogenesis is a prerequisite for tumor growth and metastasis. Vascular endothelial cell proliferation, migration, and capillary formation are stimulated by angiogenic growth factors, which include the proteins vascular endothelial growth factor, basic fibroblast growth factor, and transforming growth factor-β, and eicosanoids synthesized from n-6 fatty acids. Clinical studies have shown that angiogenesis in solid tumors relates to a poor prognosis and, in premalignant lesions, indicates potential for cancerous transformation. High-fat, n-6 fatty acid-rich diets were associated with a relatively poor prognosis in breast cancer patients; in a nude mouse model the same diet enhanced breast cancer progression, whereas n-3 fatty acids exerted suppressive effects that were associated with impaired angiogenesis. Lipoxygenase and cyclooxygenase products of n-6 fatty acid metabolism are angiogenic in in vitro assays. This activity is blocked by pharmacological inhibitors of eicosanoid biosynthesis, and one, indomethacin, suppressed n-6 fatty acid-stimulated murine mammary carcinoma growth and metastasis and tumor vascularization. Review of the experimental data suggests that selective inhibitors of eicosanoid-synthesizing enzymes and dietary intervention with n-3 fatty acids merit clinical evaluation as adjuvant therapy and chemopreventive agents.

Effects of dietary fatty acids on DU145 human prostate cancer cell growth in athymic nude mice

The purpose of this study was to examine the effects of diets containing different unsaturated fatty acids (FAs) on DU145 human prostate cancer cell growth in nude mice. In Experiment 1, groups of 25 mice were fed 23% (wt/wt) fat diets containing 18% corn oil (CO)-5% linseed oil (18:2n-6 FA-rich), 18% linseed oil (LO)-5% CO (18:3n-3 FA-rich), or 18% menhaden oil (MO)-5% CO (20:5 and 22:6n-3 FA-rich), and seven days later they were injected subcutaneously with 1 x 10(6) DU145 cells. The diets were continued for six weeks. The growth rates and final weights of tumors from the 18% CO-5% LO and 18% LO-5% CO mice were similar; there was a 30% reduction in tumor growth in the 18% MO-5% CO group (p < 0.001). The tumor phospholipid FA patterns suggested that the inhibitory effect of the high-MO diet was due, at least in part, to a reduction of arachidonic acid available for prostaglandin biosynthesis. In Experiment 2, groups of 25 mice were injected with 5 x 10(5) or 1 x 10(6) DU145 cells directly into the prostate gland and fed a high-fat linoleic acid (n-6 FA)-rich or a low-fat diet for 10 weeks. At necropsy, macroscopic cancers and microscopic intraprostatic tumors were evaluated. When the initial tumor load was 1 x 10(6) cells, all but 7 of the 50 mice had developed large macroscopic tumors; the mean tumor weight in the high-fat group was twice that in the low-fat group (p = 0.047). A stimulatory effect of dietary n-6 FA on DU145 prostate cancer cell growth may require a critical initial tumor cell mass.

Effects of Reduced Dietary Linoleic Acid Intake, Alone or Combined With an Algal Source of Docosahexaenoic Acid, on MDA-MB-231 Breast Cancer Cell Growth and Apoptosis in Nude Mice

Diets rich in linoleic acid (LA), an n-6 fatty acid, stimulate the progression of human breast cancer cell solid tumors in athymic nude mice, whereas docosahexaenoic acid (DHA) and eicosapentaenoic acid, long-chain n-3 fatty acids, exert suppressive effects. In the present study we used a novel source of DHA, in triglyceride form, to determine the effects of feeding low levels of the fatty acid on the growth of MDA-MB-231 cells injected into the thoracic mammary fat pads of female nude mice. Four different isocaloric diets were used, all of which provided 20% (wt/wt) total fat. The control diets contained 8% (20 mice) or 4% (50 mice) LA; the n-3 fatty acid-supplemented groups of 50 mice were fed 4% LA-containing diets plus 2% or 4% DHA. The tumor growth rates were reduced significantly in mice fed the 4% LA compared with the 8% LA diet; the addition of 4% DHA to the 4% LA-containing diet produced a further reduction in tumor growth rate (p < or = 0.003 at and after Week 6). The final tumor weights were also reduced in the DHA-fed mice compared with the 8% LA dietary group (2% DHA, p = 0.02; 4% DHA, p = 0.01) and in the 4% DHA-fed mice compared with the 4% LA control group (p = 0.02); a similar trend for mice fed the lower level of DHA did not achieve statistical significance. Tumor prostaglandin E2 concentrations were reduced by feeding the lower LA level; further dose-dependent decreases occurred in the DHA dietary groups and were accompanied by reduced levels of 12- and 15-hydroxyeicosatetraenoic acids. These changes in eicosanoid biosynthesis may have been responsible for the observed decreases in cell proliferation, indicated by suppressed Ki-67 expression, and increases in apoptotic activity, as reflected in TdT-mediated dUTP nick end labeling immunohistochemical staining.

The effects of a low-fat dietary intervention and tamoxifen adjuvant therapy on the serum estrogen and sex hormone-binding globulin concentrations of postmenopausal breast cancer patients.

SummaryThe purpose of the study was to determine the effect of a low-fat dietary intervention, with or without concomitant tamoxifen adjuvant therapy, on serum estrogen and sex hormone-binding globulin (SHBG) levels in postmenopausal patients with resected breast cancer. Ninety-three patients were randomized to either reduce their fat intake to 15–20% of total calories, or to a dietary control group. Serum estradiol, estrone, estrone sulfate, and SHBG concentrations were assayed at baseline, and at 6, 12, and 18 months thereafter. In 19% of patients, the preintervention serum estradiol levels were below the sensitivity of the assay (5 pg/ml). Tamoxifen had no significant effect on serum estrogen levels, but produced an elevation in SHBG. Patients with reliably quantifiable preintervention estradiol concentrations (≥ 10 pg/ml) showed a significant reduction in serum estradiol after 6 months on the low-fat diet (average, 20%; p < 0.005); this was sustained over the 18 month study period. Serum SHBG levels were increased by tamoxifen therapy, but were reduced significantly (p = 0.01) after 12 months on the low-fat diet in patients not receiving tamoxifen. No changes in serum estrone or estrone sulfate resulted from the dietary intervention. While the low-fat diet produced significant weight loss, patients treated with tamoxifen without dietary intervention showed a gain in body weight. These weight changes produced disruptions in the normal positive correlation between body weight and serum estrone sulfate, and the negative correlation with SHBG concentration.

Eicosanoids as mediators of linoleic acid-stimulated invasion and type IV collagenase production by a metastatic human breast cancer cell line.

Diets rich in linoleic acid (LA) stimulate the metastasis of MDA-MB-435 human breast cancer cells from the mammary fat pads of nude mice. This omega-6 fatty acid is metabolized to various cyclo-oxygenase and lipoxygenase products, several of which have been previously associated with tumor cell invasion and metastasis. We now report that MDA-MB-435 cells secreted increased levels of prostaglandin E2 (PGE2), and 12-hydroxyeicosatetraenoic acid (12-HETE) and 15-HETE when cultured in the presence of 2.7 µm (0.75 µg/ml) LA; 5-HETE secretion was unchanged. The 12-lipoxygenase inhibitor esculetin (20 µM) completely blocked the LA-stimulated 12-HETE secretion. Linoleic acid also increased MDA-MB-435 cell invasion in an in vitro assay; this stimulation was abolished by 20 µM esculetin, but was unaffected by piroxicam, a selective cyclooxygenase inhibitor. The effect of LA on invasion was replicated by 0.1 µM 12-HETE, but not by 5-HETE or PGE2; 15-HETE was stimulatory only at a concentration of 1.0 µm. Zymographic and Northern blot analyses showed that these events are accompanied by the induction of 92 kDa isoform type IV collagenase (metalloproteinase-9) enzymic activity and mRNA expression by exogenous LA and 12-HETE, and their suppression by the 12-lipoxygenase inhibitor. These results suggest that the effects of dietary LA on breast cancer cell metastasis in the nude mouse model are due, at least in part, to enhanced 12-HETE biosynthesis, with an associated increase in proteolytic enzyme activity and tumor cell invasiveness.

Dietary fat, fatty acids and prostate cancer

International comparisons suggest a relationship between prostate cancer incidence and dietary fat, an inference supported by migration studies, the changing incidence rates and levels of animal fat consumption in Japan and the results from some case-control studies. Overall, however, epidemiological studies have been inconclusive, and although prostate cancer is one of the hormone-dependent tumors, evidence of interactions between dietary fats and male endocrine function is incomplete. Laboratory experimentation has shown that n−6 fatty acids stimulate and n−3 fatty acids inhibit human prostate cancer cells in culture; also, feeding diets rich in marine oils suppresses growth of these cells as solid tumors in athymic nude mice. These growth effects of polyunsaturated fatty acids appear to involve both prostaglandins and leukotrienes and to interconnect with autocrine regulation by epidermal growth factor-related polypeptides.

Stimulation of growth of human breast cancer cell lines in culture by linoleic acid

Linoleic acid, an omega-6 unsaturated fatty acid, stimulated growth of the MDA-MB-231 and MCF-7 human breast cancer cell lines in culture. Responses of the estrogen-independent MDA-MB-231 cells both in serum-free medium and with 1% fetal bovine serum added were positively correlated with linoleic acid concentration over the entire range examined (5-750 ng/ml). Growth stimulation of the estrogen-responsive MCF-7 cell line was maximal at a LA concentration of 500 ng/ml when cultured in 1% fetal bovine serum-containing medium with added estradiol. Linoleic acid had no mitogenic effect on three human cancer cell lines derived from sites other than breast, or on untransformed 3T3 cells.

Epidermal growth factor-like proteins in breast fluid and human milk

Epidermal growth factor (EGF), and the transforming growth factor-alpha (TGF-alpha) family of proteins, which also bind to the EGF receptor, have been associated with human breast cancer. The total EGF-like proteins were determined by a radioreceptor assay, and TGF-alpha by radioimmunoassay, in human milk and breast fluid samples. The breast fluids were collected by nipple aspiration from healthy premenopausal women. Both the 24 milks and 18 breast fluids assayed contained EGF-like proteins, at concentrations ranging from 32-600 ng/ml (median, 140 ng/ml), and 62-654 ng/ml (median, 205 ng/ml) respectively. Immunoreactive TGF-alpha proteins were detected at higher levels in 21 breast fluids (range, 0-50.0; median 5.1 ng/ml) than in 24 milk samples (range, 0-8.4; median, 0.8 ng/ml).

Enhanced angiogenesis and growth of 12-lipoxygenase gene-transfected MCF-7 human breast cancer cells in athymic nude mice.

Transfection of the estrogen-dependent and poorly invasive MCF-7 human breast cancer cell line so that it stably overexpressed 12-lipoxygenase and secreted high levels of 12-hydroxyeicosatetraenoic acid when cultured with arachidonate resulted in rapid growth in athymic nude mice when compared with the parental line. This enhanced acquisition of tumor mass was a result of both increased cell proliferation and reduced apoptotic cell death and was accompanied by high angiogenic activity.