Modulation of MCF-7 breast cancer cell signal transduction by linoleic acid and conjugated linoleic acid in culture.

The effects of modifying membrane fatty acid composition on cell growth, phospholipase C (PLC) and protein kinase C (PKC) activities, and prostaglandin E2 (PGE2) secretion were investigated. Hormone responsive MCF-7 human breast cancer cells were incubated in a serum-free medium containing epidermal growth factor and supplemented with physiologic concentrations (0.18-1.78 x 10(-5) M) of linoleic acid (LA) or conjugated linoleic acid (CLA). Linoleic acid stimulated cancer cell growth, while CLA was inhibitory. Supplementation with LA or CLA altered cell membrane composition. Linoleic acid stimulated PLC activity with or without GTP gamma (S), and tended to increase membrane PKC activity. However, CLA supplementation did not modify membrane PLC or PKC activity. Prostaglandin E2 secretion was not influenced by LA or CLA. These data show that growth inhibition by CLA was not mediated through PLC-, PKC- or PGE2-dependent signal transduction pathways, suggesting that another inhibitory mechanism may be involved. Although biological differences appeared to be modest (5-20% of control), the fact that LA and CLA treatment resulted in significant biological effects at physiologic concentrations is relevant, since most human cancers require years to develop.

Safflower oil consumption does not increase plasma conjugated linoleic acid concentrations in humans.

Conjugated linoleic acid (CLA) is a mixture of positional and geometric isomers of linoleic acid (LA) with conjugated double bonds. CLA has anticarcinogenic properties and has been identified in human tissues, dairy products, meats, and certain vegetable oils. A variety of animal products are good sources of CLA, but plant oils contain much less. However, plant oils are a rich source of LA, which may be isomerized to CLA by intestinal microorganisms in humans. To investigate the effect of triacylglycerol-esterified LA consumption on plasma concentrations of esterified CLA in total lipids, a dietary intervention (6 wk) was conducted with six men and six women. During the intervention period a salad dressing containing 21 g safflower oil providing 16 g LA/d was added to the subjects' daily diets. Three-day diet records and fasting blood were obtained initially and during dietary and postdietary intervention periods. Although LA intake increased significantly during the dietary intervention, plasma CLA concentrations were not affected. Plasma total cholesterol and LDL-cholesterol concentrations were significantly lower after addition of safflower oil to the diet. In summary, consumption of triacylglycerol-esterified LA in safflower oil did not increase plasma concentrations of esterified CLA in total lipids.

Proliferative responses of normal human mammary and MCF-7 breast cancer cells to linoleic acid, conjugated linoleic acid and eicosanoid synthesis inhibitors in culture.

Potential mechanisms for the stimulation or inhibition of cell growth by linoleic acid (LA) and conjugated linoleic acid (CLA) were investigated by using eicosanoid synthesis inhibitors. Normal human mammary epithelial cells (HMEC) and MCF-7 breast cancer cells were incubated in serum-free medium supplemented with LA or CLA and cyclooxygenase (indomethacin; INDO) or lipoxygenase (nordihydroguaiaretic acid; NDGA) inhibitors. Linoleic acid stimulated the growth and [3H]thymidine incorporation of normal HMEC and MCF-7 cancer cells, while CLA was inhibitory. Supplementation with LA increased intracellular lipid peroxide concentrations in normal HMEC and MCF-7 cancer cells, whereas CLA did not affect lipid peroxide formation. Normal HMEC and MCF-7 cells supplemented with LA and INDO or NDGA resulted in growth inhibition. The treatment of normal HMEC with CLA and INDO or NDGA, and MCF-7 cells with CLA and INDO stimulated cell growth. However, the addition of CLA and NDGA to MCF-7 cells resulted in synergistic growth suppression suggesting that CLA effects were mediated through lipoxygenase inhibition. Although NDGA was more inhibitory of cell growth in the presence of LA or CLA than INDO, growth was associated with both prostaglandin and leukotriene production. Additional studies are warranted to elucidate the mechanism(s) whereby LA or CLA affect breast cell growth.

Differential stimulatory and inhibitory responses of human MCF-7 breast cancer cells to linoleic acid and conjugated linoleic acid in culture.

Consumption of dietary fat has been linked to the high incidence of certain cancers. However, recent research has stimulated interest in conjugated linoleic acid (CLA), a newly recognized anticarcinogenic fatty acid. Human MCF-7 breast cancer cells were incubated for 12 d in culture medium supplemented with various concentrations (1.78-7.14 x 10(-5) M) of linoleic acid (LA) or CLA. Linoleic acid initially stimulated MCF-7 cell growth with an optimal effect at concentrations of 3.57-7.14 x 10(-5) M, but was inhibitory at similar concentrations after 8 and 12 d of incubation. In contrast, CLA was inhibitory to cancer cell growth at all concentrations and times tested. Cell growth inhibition by CLA was dose- and time-dependent. Growth retardation at the prescribed LA and CLA concentrations ranged, respectively, from 4 to 33% and 54 to 100% following 8 to 12 d of treatment. At similar LA and CLA concentrations, cytostatic and cytotoxic effects of CLA were more pronounced (8-81%) than LA. These in vitro results suggest that CLA is cytotoxic to MCF-7 cells.

Inhibitory effect of conjugated dienoic derivatives of linoleic acid and beta-carotene on the in vitro growth of human cancer cells.

The effects of physiologic concentrations of conjugated linoleic acid (CLA) and beta-carotene were assessed on human (M21-HPB, malignant melanoma; HT-29, colorectal; MCF-7, breast) cancer cells. The incubation of cancer cells with CLA showed significant reductions in proliferation (18-100%) compared to control cultures. M21-HPB and MCF-7 cell mortality was dose- and time-dependent. beta-Carotene was inhibitory to breast cells only. MCF-7 cells supplemented with CLA incorporated significantly less [3H]leucine (45%), [3H]uridine (63%) and [3H]thymidine (46%) than control cultures. M21-HPB and HT-29 cells supplemented with CLA incorporated less [3H]leucine (25-30%). These in vitro results suggest that CLA and beta-carotene may be cytotoxic to human cancer cells in vivo.

Evaluation of cancer patient leukocyte responses in the presence of physiologic and pharmacologic pyridoxine and pyridoxal levels.

Peripheral blood samples from six cancer patients (five colon cancer, one lung cancer) and six healthy volunteers were tested in vitro for oxygen radical production by phagocytic cells and in assays of mitogen-induced lymphoblastogenesis at physiologic and pharmacologic concentrations of pyridoxine (PN, 1.8-96 nmol/ml) or pyridoxal (PL, 0.08-90 nmol/ml). Plasma levels of pyridoxal-5'-phosphate (PLP), 4-pyridoxic acid (4PA), pyridoxamine phosphate (PMP), and PL were also determined. Phagocytic cells from three patients showed significantly increased capacity for oxygen radical production when incubated in PL-, but not PN-supplemented media. Oxygen burst capacity of cells from healthy subjects was significantly enhanced by PN-, but not PL-enriched media. Lymphocyte responsiveness to phytohemagglutinin or pokeweed mitogen (PWM) stimulation showed a modest increase in cell activation in three patients as the concentration of PN was increased; with concanavalin A, two showed enhanced responsiveness. On the other hand, PL-supplementation resulted in greater cell proliferation only with PWM. The cancer patients had significantly lower plasma PLP, 4PA, and PMP levels when compared with the healthy volunteers. These data indicate that in the cancer patients and in a majority of the healthy volunteers, vitamin B-6 status was marginal or deficient and suggest that increasing PN or PL in vivo levels may augment functions related to cell-mediated immunity.

Effect of pyridoxine and pyridoxal on the in vitro growth of human malignant melanoma.

The in vitro effect of vitamin B-6 supplementation on the growth of a human malignant melanoma cell line (M21-HPB) was investigated. Varying concentrations of pyridoxine (PN) or pyridoxal (PL) were added to cell cultures and incubated for 12 days. Pharmacologic levels of PL (0.25-0.5 mM) resulted in significant reductions in cell proliferation. Physiologic levels (0.005 mM) did not retard growth. Cells incubated with PN showed growth stimulation. Intracellular levels of PL and pyridoxal 5'-phosphate (PLP) were increased in cells exposed to pharmacologic PL (0.05-0.5 mM) concentrations, but not PN. These data suggest that PL or PLP may be involved in regulating the growth of melanoma cells and that vitamin B-6 may have potential use as an antineoplastic agent.

Suppression of tumor growth and enhancement of immune status with high levels of dietary vitamin B6 in BALB/c mice.

Effects of dietary vitamin B6 at levels ranging from deficiency to megadoses on the development of herpes simplex virus type 2-transformed (H238) cell-induced tumors and on in vitro responses relating to cell-mediated immunity were examined. Male BALB/cByJ mice (n = 260), 5 weeks of age, were fed 20% casein diets containing pyridoxine (PN) at 0.2, 1.2 for the control diet, 7.7, or 74.3 mg/kg diet for 4-11 weeks. After 4 weeks of dietary treatment, 120 of the mice received an injection of H238 cells; mice without H238 injection served as controls. At 4, 8, and 11 weeks, animals from each group were euthanized and blood and spleen samples obtained. Mice fed 0.2 mg PN developed mild deficiency symptoms and gained significantly less weight than those fed 1.2-, 7.7-, and 74.3-mg PN diets. Thirteen to 16 days after tumor cell injection, primary tumor incidence was lowest in mice fed 74.3 mg PN; later, incidence among groups was similar. Mice fed 1.2 mg PN had the largest primary tumor volume, the highest incidence of lung metastases, and the greatest number of metastatic nodules per animal at 7 weeks post injection. Overall, lower tumor volumes were found in animals fed 7.7 and 74.3 mg PN (14 and 32% less than the tumor volume for those fed 1.2 mg PN, respectively); mice fed 0.2 mg PN had the lowest tumor volume. Blood and spleen lymphoproliferative response to stimulation by phytohemagglutinin or concanavalin A generally tended to be higher in mice fed 7.7 and 74.3 mg PN as compared to that in animals fed either 0.2 or 1.2 mg PN. However, decreased mitogen-stimulated responsiveness was observed in all animals with progressive tumor growth. Tumor growth also resulted in splenomegaly and increased thymic atrophy. Significant negative relationships between tumor volume and tumor pyridoxal 5-phosphate (PLP) concentrations were observed for 1.2-, 7.7-, and 74.3-mg PN diet groups. These data suggest that high dietary intake of vitamin B6 may have suppressed tumor development by either immune enhancement or PLP growth regulation of this tumor.

Selenium status of vegeterians, nonvegetarians, and hormone-dependent cancer subjects.

Human blood selenium (Se) levels have been related to the types of food consumed, bioavailability of Se, and various disease states, including cancer. Some of these interrelationships were investigated in this study in Corvallis, OR (a low soil-Se region) using adult vegetarian and omnivorous subjects, some of whom had hormone-dependent cancer. The study groups were comprised of 48 Seventh-day Adventist vegetarians, 16 Seventh-day Adventist nonvegetarians, 52 non-Seventh-day Adventist nonvegetarians, and 16 nonvegetarian hormone-dependent cancer subjects. Fasting blood samples and 3-days dietary intake information were obtained from each subject. Whole blood Se levels, measured fluorimetrically, correlated positively with dietary protein, riboflavin, niacin, and oleic and linoleic acids but not with 11 other nutrients. Due to limited literature values, dietary Se could not be assessed. There was no significant difference in blood Se values between the four groups (which ranged from 0.069 microgram Se/ml for Seventh-day Adventist nonvegetariants to 0.112 +/- 0.050 microgram Se/ml for non-Seventh-day Adventists nonvegetarians and nonvegetarian hormone-dependent cancer patients). All values were well below averages reported for other regions of the United States. These data suggest a relationship between blood Se and the consumption of meat, milk, and cereal products, but it is not simply a difference between the vegetarian and nonvegetarian diets.

Effect of high dose ascorbic acid on vitamin B6 metabolism.

The influence of ascorbic acid intake on vitamin B6 metabolism in humans was determined in three separate studies. Five subjects in study I and two subjects in study II received 0.5 ( 2 days) and 1 g (7 days) doses of L-ascorbic acid (AA), respectively, before supplementation with 2 mg of pyridoxine hydrochloride (PN-HCl). From daily diet records the percentage of the intake of vitamin B6 excreted as urinary 4-pyridoxic acid (4PA) was calculated. Because of the variability in vitamin B6 intake, study III was conducted with four male and four female subjects, who received identical meals on days 2, 3, 9, and 10 and 1 g of AA on days 4 to 10. Two milligrams of PN.HCl were given the day before and the last day of AA administration. In contrast to studies I and II, where excretion of 4PA appeared to increase on the day of PN.HCl supplementation plus AA compared to pre-AA, urinary 4PA excretion in the eight subjects of study III was not significantly different when the same PN.HCl supplemented pre-AA and post-AA days were compared. Also, the fasting plasma pyridoxal 5'-phosphate level was not significantly altered when AA was administered. It is concluded that short-term AA supplementation did not alter vitamin B6 metabolism.