Najla Guthrie

Inhibition of human breast cancer cell proliferation and delay of mammary tumorigenesis by flavonoids and citrus juices

Two citrus flavonoids, hesperetin and naringenin, found in oranges and grapefruit, respectively, and four noncitrus flavonoids, baicalein, galangin, genistein, and quercetin, were tested singly and in one-to-one combinations for their effects on proliferation and growth of a human breast carcinoma cell line, MDA-MB-435. The concentration at which cell proliferation was inhibited by 50% (IC50), based on incorporation of [3H]thymidine, varied from 5.9 to 140 micrograms/ml for the single flavonoids, with the most potent being baicalein. IC50 values for the one-to-one combinations ranged from 4.7 micrograms/ml (quercetin + hesperetin, quercetin + naringenin) to 22.5 micrograms/ml (naringenin + hesperetin). All the flavonoids showed low cytotoxicity (> 500 micrograms/ml for 50% cell death). Naringenin is present in grapefruit mainly as its glycosylated form, naringin. These compounds, as well as grapefruit and orange juice concentrates, were tested for their ability to inhibit development of mammary tumors induced by 7,12-dimethylbenz[a]anthracene (DMBA) in female Sprague-Dawley rats. Two experiments were conducted in which groups of 21 rats were fed a semipurified diet containing 5% corn oil and were given a 5-mg dose of DMBA intragastrically at approximately 50 days of age while in diestrus. One week later, individual groups were given double-strength grapefruit juice or orange juice or fed naringin or naringenin at levels comparable to that provided by the grapefruit juice; in the second experiment, the rats were fed a semipurified diet containing 20% corn oil at that time. As expected, rats fed the high-fat diet developed more tumors than rats fed the low-fat diet, but in both experiments tumor development was delayed in the groups given orange juice or fed the naringin-supplemented diet compared with the other three groups. Although tumor incidence and tumor burden (grams of tumor/rat) were somewhat variable in the different groups, rats given orange juice had a smaller tumor burden than controls, although they grew better than any of the other groups. These experiments provide evidence of anticancer properties of orange juice and indicate that citrus flavonoids are effective inhibitors of human breast cancer cell proliferation in vitro, especially when paired with quercetin, which is widely distributed in other foods.

Inhibition of proliferation of estrogen receptor-positive MCF-7 human breast cancer cells by flavonoids in the presence and absence of excess estrogen

The flavonoids are a group of naturally-occurring, low molecular weight compounds that are widespread in plants. Representatives of several different classes of flavonoids were tested for their effects on the proliferation of an estrogen receptor-positive human breast cancer cell line, MCF-7. The IC50S (concentration at which cell proliferation was inhibited by 50%), based on [3H]thymidine incorporation, ranged from 4.2 to 18.0 micrograms/mL. The cells were viable at these concentrations. The possibility that flavonoids may block cell proliferation by binding to the estrogen receptor was explored. The cells were depleted of endogenous steroids and incubated with individual flavonoids at their IC50 concentration. Half of the cells were exposed to an excess concentration of 17 beta-estradiol to see if this affected antiproliferation by the flavonoids. Of the flavonoids tested, only the inhibition of cell proliferation by genistein was reversed with the addition of excess, competing estrogen. Baicalein, galangin, hesperetin, naringenin and quercetin apparently exert their antiproliferative activity via some other mechanism.

Effect of tocotrienols on the growth of a human breast cancer cell line in culture.

The tocotrienol-rich fraction (TRF) of palm oil consists of tocotrienols and some α-tocopherol (α-T). Tocotrienols are a form of vitamin E having an unsaturated side-chain, rather than the saturated side-chain of the more common tocopherols. Because palm oil has been shown not to promote chemically-induced mammary carcinogenesis, we tested effects of TRF and α-T on the proliferation, growth, and plating efficiency (PE) of MDA-MB-435 estrogen-receptor-negative human breast cancer cells. TRF inhibited the proliferation of these cells with a concentration required to inhibit cell proliferation by 50% of 180 μg/mL, whereas α-T had no effect at concentrations up to 1000 μg/mL as measured by incorporation of [3H]thymidine. The effects of TRF and α-T also were tested in longer-term growth experiments, using concentrations of 180 and 500 μg/mL. We found that TRF inhibited the growth of these cells by 50%, whereas α-T did not. Their effect on the ability of these cells to form colonies also was studied, and it was found that TRF inhibited PE, whereas α-T had no effect. These results suggest that the inhibition is due to the presence of tocotrienols in TRF rather than α-T.

Specific versus non-specific effects of dietary fat on carcinogenesis.

It will be apparent from this review that dietary fat can exert both specific and non-specific effects on carcinogenesis, at least in experimental animals. The non-specific effects appear to be related primarily to effects of dietary fat on energy balance. Although a positive energy balance can be achieved on a high-carbohydrate low-fat diet, it is much more likely to occur on a high-fat diet because of the high energy density of fat [101] and the fact that dietary fat is less capable of imparting a sense of satiety [102]. A continuing state of positive energy balance leads to obesity which has been associated with increased risk of cancer at a number of sites, including endometrium [103-106], postmenopausal breast cancer [107-113], renal cancer [114,115] and possibly cancers of the colorectum [116-122], pancreas [103,123] and prostate [124]. Whereas the non-specific effects of dietary fat appear to be deleterious for cancer, the specific effects in some cases can be beneficial. Examples are long-chain n-3 polyunsaturated fatty acids. CLA and tocotrienols. It is still too early to predict whether these may be of value in the prevention and/or treatment of human cancer but they seem worthy of further investigation. Knowledge of their mechanism of action may suggest novel approaches to the cancer problem and, as in the case of vitamins A and D, it may be possible to find analogues with more potent anti-cancer activity.

Famesylamine: An inhibitor of famesylation and growth ofras-transformed cells

Farnesylamine, an analogue of farnesol, was shown to inhibit growth of PAP2 cells (ras-transformed NIH 3T3 cells) in a dose-dependent manner. This inhibition was overcome by adding farnesol to the culture medium, but not by adding geranylgeraniol, squalene, cholesterol, dolichol, myristic acid or palmitic acid. Farnesylamine inhibited both farnesyl/protein transferase and geranylgeranyl/protein transferase in whole cell extracts and also inhibited the prenylation of proteins, particularlyras p21, in PAP2 cells. Inhibition of prenylation was associated with increased biosynthesis of other products of the mevalonate biosynthetic pathway. These observations suggest that inhibition of the growth of PAP2 cells by farnesylamine may be due to blocking ofras-mediated signal transduction. This offers a means of investigating mechanisms involved inras action and raises the possibility of developing novel strategies for anticancer therapy.

Effects of long-chain fatty amines on the growth of ras-transformed NIH 3T3 cells

A number of aliphatic primary amines were tested for their effects on the growth of ras-transformed NIH 3T3 cells (PAP2 cells), as measured by incorporation of tritiated thymidine into DNA. Long-chain, saturated amines (C12 to C18) were growth inhibitory, whereas short-chain amines (C6, C8) were not. Farnesylamine, a branched-chain, unsaturated amine (C15), had an IC50 of 6.9 microM compared to IC50 values of 13.1 to 45.8 microM for straight-chain, saturated amines. Oleylamine, with an IC50 of 0.1 microM, was the most potent inhibitor. The long-chain amines, but not the short-chain amines, were also effective inhibitors of protein kinase C, assayed in vitro in a cell-free system. In addition, studies with indo-1-loaded PAP2 cells showed that long-chain amines induced a reversible rise in intracellular free Ca2+ concentration. Growth inhibition by the amines was positively correlated with this effect, suggesting that factors other than protein kinase C may be involved in the inhibition of growth of PAP2 cells by long-chain amines.

Effects of dietary minerals on cholesterol metabolism in rabbits fed cholesterol-free atherogenic diets

Abstract To investigate the effect of dietary minerals on cholesterol metabolism, four groups of rabbits were fed semipurified diets with modified mineral mixes for 7 weeks. Compared with the control diet that contained minerals at the 4% (wt/wt) level (diet A), LDL-cholesterol was significantly higher (P