Kenneth K. Carroll

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.

Effects of level and type of dietary fat on incidence of mammary tumors induced in female Sprague-Dawley rats by 7, 12-dimethylbenz(α)anthracene.

Female Sprague-Dawley rats on semisynthetic diets containing 10% and 20% by weight of corn oil developed more mammary adenocarcinomas after treatment with a single oral dose of 7,12-dimethylbenz(α)anthracene than similar rats on diets containing only 0.5% or 5% corn oil. Experiments with 10 different fats and oils fed at the 20% level indicated that unsaturated fats enhance the yield of adenocarcinomas more than saturated fats. Fibroadenomas and adenomas were also found in small numbers in all dietary groups but the yield did not seem to be influenced by level or type of dietary fat. The possible relevance of these findings to the incidence of breast cancer in humans is discussed.

Dietary polyunsaturated fat in relation to mammary carcinogenesis in rats

High fat diets promote the development of mammary tumors induced in rats by 7,12-dimethylbenz(a)anthracene (DMBA), and polyunsaturated fats are more effective than saturated fats. This difference is related to the linoleic acid content of polyunsaturated vegetable oils, but the amount of linolealte required for maximum tumor promotion appears to be higher than indicated by earlier experiments. Comparison of the effects of a polyunsaturated vegetable oil (corn oil) containing linoleate with a fish oilo (menhaden oil) containing polyunsaturated fatty acids derived from linolenic acid showed that higher dietary mammary tumors, while corresponding levels of menhaden oil had an inhibitory effect. This is further evidence that promotion of mammary tumorigenesis by polyunsaturated vegetable oils may be mediated by prostaglandins or other biologically active eicosanoids derived from n−6 fatty acids.

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.

Obesity as a risk factor for certain types of cancer

Summary and ConclusionsIn conclusion, obesity has been associated with increased risk for a number of different types of cancer. The evidence has been most consistent for endometrial cancer, breast cancer in postmenopausal women, and renal cell cancer. More variable results have been reported for colorectal, prostate and pancreatic cancer.Possible mechanisms by which obesity may influence cancer risk include alteration in hormonal patterns, including sex hormones and insulin, and factors such as the distribution of body fat and changes in adiposity at different ages. The increasing prevalence of obesity in many parts of the world emphasizes the importance of learning more about the relationship between obesity and cancer and the mechanisms involved in their interaction.

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.

Regulation of HepG2 cell apolipoprotein B metabolism by the citrus flavanones hesperetin and naringenin

Our previous studies showed that replacing the drinking water of rabbits fed a casein-containing diet with either orange juice or grapefruit juice reduced serum low density lipoprotein cholesterol and hepatic cholesteryl ester concentrations. To determine whether the changes observed in rabbits were due to flavonoids present in the juices acting directly on the liver’ the effects of hesperetin and naringenin on net apolipoprotein B (apoB) secretion by HepG2 cells were investigated. These flavanones dose-dependently reduced net apoB secretion by up to 81% after a 24 h incubation’ while doses of 60 μg/mL reduced net apoB secretion by 50% after 4 h. Coincubation with the proteasome inhibitor’ MG-132’ did not alter the ability of the flavonoids to reduce net apoB secretion over 4 h’ suggesting that the flavonoid-induced changes in apoB metabolism were not due to a direct increase in proteasomal activity. However’ the flavonoids were unable to reduce net apoB secretion after 4 h in the presence of oleate’ suggesting that these compounds may interfere with the availability of neutral lipids for lipoprotein assembly. Furthermore’ our 14C-acetatelabeling studies showed a 50% reduction in cholesteryl ester synthesis in the presence of either flavonoid’ which could account for the reduction in net apoB secretion caused by incubation with these compounds. These in vitro studies suggest that hesperetin and naringenin may’ in part’ reduce net apoB secretion by HepG2 cells by inhibiting cholesteryl ester synthesis and that these compounds are good candidates for further in vivo studies to determine whether they are responsible for the cholesterol-lowering properties of dietary citrus juices.

Plasma cholesterol levels in rabbits fed low fat, cholesterol-free, semipurified diets: effects of dietary proteins, protein hydrolysates and amino acid mixtures

Abstract Rabbits become hypercholesterolemic when fed a low fat, cholesterol-free, semisynthetic diet containing casein as the dietary protein. This did not occur when the casein was replaced by soy protein isolate or any one of seven other plant protein preparations. Doubling the amounts of either the casein or soy protein isolate from 25 to 50% by weight of the diet, made no significant difference to their effects on plasma cholesterol. Soy protein isolate was effective in counteracting the hypercholesterolemic response to casein when mixtures of the two proteins were fed. There appeared to be no relationship between body weight gains and plasma cholesterol levels in rabbits fed the different diets. Animals fed the higher level of casein failed to gain weight, whereas growth was not significantly impaired by doubling the level of soy protein isolate in the diet. Better growth was obtained with mixtures of casein and soy protein isolate than with either protein alone. An enzymatic hydrolysate of casein or a mixture of L-amino acids equivalent to casein gave elevated plasma cholesterol levels similar to those obtained with the intact protein. Plasma cholesterol levels remained low in rabbits fed an enzymatic digest of soy protein. A moderate, but not significant, increase in plasma cholesterol was observed when a mixture of L-amino acids equivalent to soy protein isolate was fed. The results of these experiments indicate that the level of plasma cholesterol can be influenced by the amino acids supplied in the diet.

Distribution, metabolism and function of dolichol and polyprenols

Polyisoprenoid alcohols consisting of 9 or more isoprene units are present in all living cells. They can be fully unsaturated (polyprenols) or alpha-saturated (dolichol). Dolichol forms may have additional saturation at or near the omega-end. Some species contain ony dolichol or only polyprenols while others have nearly equal amounts of both types. Some polyisoprenoid alcohols consist entirely of trans isoprene units but most, including dolichol, contain both trans and cis units. Considerable advances in lipid methodology have occurred since the first review of polyisoprenoid alcohols by Hemming in 1974. For example, direct analysis of both dolichol and Dol-P by HPLC has replaced earlier methods which were often both insensitive and inaccurate. The availability of radiolabeled dolichol and polyprenols has facilitated studies concerning the metabolism and distribution of these compounds. Those studies suggest that only a small portion of the dolichol present in cells is likely to be involved in glycosylation. Polyisoprenoid alcohols are usually present at a family of homologues where each differs in size by one isoprene unit. Little or no size related specificity has been observed for any reaction involving dolichol or polyisoprenol intermediates. The overall length of polyisoprenoid alcohols may, however, affect the manner in which these compounds influence the physical and biochemical properties of membranes. Studies on the biosynthetic pathway leading from cis, trans Pol-PP by phosphatase action. The formation of the dolichol backbone from a polyprenol requires the action of an additional enzyme, an alpha-saturase. This enzyme does not always act at the level of a single common substrate, since Pol-PP, Pol-P, and polyprenol all appear to be utilized as substrates. The major product of the de novo pathway differs among different species. Dol-P would appear to be the most energy efficient end-product since it can participate directly in glycoprotein formation. Most often, however, Dol-P is not the major product of metabolic labeling experiments. In some cases, dolichol is formed so that rephosphorylation is required to provide Dol-P for participation in glycoprotein formation. The kinase responsible for this phosphorylation appears to bypass the considerable stores of dolichol present in tissues (i.e. sea urchin eggs) in favor of dolichol derived directly from de novo synthesis. Although HMGR is a major regulatory component of the pathway leading to polyisoprenoid alcohols and cholesterol, control is most often not co-ordinated.(ABSTRACT TRUNCATED AT 400 WORDS)

Dietary polyunsaturated fat versus saturated fat in relation to mammary carcinogenesis

High levels of dietary fat have been shown to promote the development of mammary tumors induced in rats by 7,12-dimethylbenz(α)anthracene, and polyunsaturated fats were found to be more effective than saturated fats. In further studies it was found that diets containing 3% sunflowerseed oil (polyunsaturated fat) and 17% beef tallow or coconut oil (saturated fats) enhance tumorigenesis as much as a diet containing 20% sunflowerseed oil. Rats on these diets developed at least twice as many tumors as those fed diets containing either 3% sunflowerseed oil or 20% of the saturated fats alone. These results are in accord with human epidemiological data which show that breast cancer mortality in different countries is positively correlated with total fat intake but not with intake of polyunsaturated fat. Total fat intake varies greatly in different countries, but most human diets probably contain levels of polyunsaturated fat at least equivalent to 3% sunflowerseed oil.