Inhibition of murine bladder tumorigenesis by soy isoflavones via alterations in the cell cycle, apoptosis, and angiogenesis.

Soy isoflavones exhibit a number of biological effects, suggesting that they may have a role in cancer prevention. Our objectives are to determine whether components of soy products or purified soy isoflavones can inhibit the progression of bladder cancer. We compared the in vitro effects of pure soy isoflavones and soy phytochemical concentrate on growth curves, cell cycle progression, and apoptosis in murine and human bladder cancer cell lines. Pure soy isoflavones (genistein, genistin, daidzein, and biochanin A) and soy phytochemical concentrate exhibit dose-dependent growth inhibition of murine (MB49 and MBT-2) and human (HT-1376, UM-UC-3, RT-4, J82, and TCCSUP) bladder cancer cell lines, although the degree of inhibition varies among lines. Soy isoflavones induce a G2-M cell cycle arrest in all human and murine lines evaluated by flow cytometry. In addition, some bladder cancer lines show DNA fragmentation consistent with apoptosis. We next evaluated the ability of genistein, soy phytochemical concentrate, and soy protein isolate, respectively, to inhibit the growth of transplantable murine bladder cancer in vivo. C57BL/6 mice were randomly assigned to treatment groups (n = 12/group): (a) AIN-76A diet; (b) AIN-76A diet plus genistein, i.p., 50 mg/kg body weight/day; (c) AIN-76 diet with soy phytochemical concentrate at 0.2% of the diet; (d) AIN-76 diet with soy phytochemical concentrate at 1.0% of the diet; and (e) AIN-76A diet with soy protein isolate, 20% by weight. Mice were inoculated s.c. with 5 x 10(4) syngeneic MB49 bladder carcinoma cells, and tumor growth was quantitated. Neither genistein nor soy products reduced body weight gain. Tumor volumes from mice treated with genistein, dietary soy phytochemical concentrate at 1%, or dietary soy protein isolate were reduced by 40% (P < 0.007), 48% (P < 0.001), or 37% (P < 0.01), respectively, compared with controls. We characterized the effects of treatment on several biomarkers in tumor tissue: proliferation index by proliferating cell nuclear antigen staining, apoptotic index by terminal deoxynucleotidyltransferase-mediated dUTP-biotin nick end labeling staining, and angiogenesis by microvessel quantitation. Soy products reduced angiogenesis, increased apoptosis, and slightly reduced proliferation while showing no histopathological effects on the normal bladder mucosa. Our data suggest that soy isoflavones can inhibit bladder tumor growth through a combination of direct effects on tumor cells and indirect effects on the tumor neovasculature. Soy products warrant further investigation in bladder cancer prevention and treatment programs or as antiangiogenic agents.

Intracellular beta-carotene transport in bovine liver and intestine is not mediated by cytosolic proteins.

The mechanisms responsible for the movement of absorbed carotenoids between intracellular organelles and for the incorporation of carotenoids into serum lipoproteins are unknown. It was the objective of this study to use bovine liver and intestine as a model to study the possible cytosolic protein-mediated carotenoid transfer between liposomes and mitochondria in vitro. Liposomes containing beta-[3H]carotene were incubated with isolated bovine hepatic mitochondria (1 mg protein) and various quantities of liver cytosol or intestinal mucosal cell cytosol (0-6 mg protein) for up to 1 h at 37 degrees C. The beta-[3H]carotene activity in liposomes was then measured over time to determine percent transfer of beta-carotene to mitochondria. Over the time period studied, the rate of beta-carotene transfer was unaffected by increasing levels of either hepatic or intestinal cytosolic protein. These results suggest that unlike many other lipid species, intracellular transport of beta-carotene is not mediated by cytosolic transport proteins and must occur by other mechanisms such as vesicular transport or by membrane-bound proteins.

Brush border membrane vesicles from rats and gerbils can be utilized to evaluate the intestinal uptake of all-trans and 9-cis beta-carotene.

Intestinal brush border membrane vesicles (BBMV) have previously been used to investigate the absorptive processes of a wide variety of nutrients. The first objective of this work was to develop a BBMV system to study carotenoid uptake using rat small intestine. The uptake of all-trans beta-carotene by rat BBMV was linear up to 5.9 micromol/L, suggesting passive uptake at the concentrations used in this study. Both all-trans beta-carotene and 9-cis beta-carotene were taken up at a similar rate by the rat BBMV. The vesicle system was then utilized to investigate the uptake of retinol and beta-carotene by BBMV isolated from Mongolian gerbils which had been subjected to three different vitamin A depletion and refeeding protocols. Three groups of gerbils (n = 10/group) were fed vitamin A-deficient diets for 8 wk. One group was killed at that point (marginally depleted), a second group was refed vitamin A for 5 d and a third group was further depleted of vitamin A for 5 d. Pooled BBMV from the marginally depleted group demonstrated greater uptake of all-trans beta-carotene than pooled BBMV from the other two groups of gerbils while those from the group refed vitamin A for 5 d had greater uptake than the group that was depleted for 5 additional days (P < 0.01). In contrast, the uptake of retinol by BBMV was not significantly different among the three groups of gerbils. The results of the gerbil study suggest that as they become increasingly vitamin A deficient, impairment of all-trans beta-carotene uptake by intestinal mucosal cells may be taking place. The BBMV system should prove valuable for the evaluation of the intestinal mucosal cell membrane uptake of carotenoids and retinoids under various nutritional conditions.

The crystalline form of carotenes and the food matrix in carrot root decrease the relative bioavailability of beta- and alpha-carotene in the ferret model.

OBJECTIVE: The objective of this study was to investigate the relative bioavailability (BV) of beta-carotene (BC) and alpha-carotene (AC) from different extracts of carrots utilizing the ferret model. METHODS: Five groups of animals (n = 6) were provided free access to a carotenoid-free, vitamin A-adequate diet and tap water for 2 weeks, then for 3 consecutive days 10 mg of BC was provided in 30--40 mL of one of the following fluids as partial replacement for tap water along with diet: 1) commercial BC beadlets dispersed in distilled water (control), 2) non-heated carrot juice, 3) heated carrot juice, 4) non-heated isolated carrot chromoplasts, or 5) heated carrot chromoplasts. The animals were killed and samples of blood and tissues (liver, adrenal, lung, kidney, spleen) were collected and analyzed for AC and BC concentrations. RESULTS: The tissue analysis of BC concentrations indicated that BC-beadlet-supplemented animals had significantly higher BC concentrations than other groups (p<0.01). Carrot chromoplast-supplemented animals had significantly higher tissue BC and AC concentrations than carrot juice-supplemented animals. Heat treatment tended to reduce the relative BV of carotenoids but the differences between heated and non-heated juices or isolated carrot chromoplasts were not significantly different. CONCLUSIONS: These results suggest that the food matrix, probably pectin-like fibers, and the crystalline form of carotenoids in carrot chromoplast are the primary factors that reduce the relative BV of carotenoids from carrot juice.

Beta-carotene uptake and tissue distribution in ferrets (Mustela putorius furo).

Ferrets accumulate beta-carotene in liver and adipose tissue after chronic feeding. This study was designed to further evaluate the time course of uptake and depletion of beta-carotene in ferret serum and tissues. Male ferrets (n = 15; 1000-1200 g) were given a single dose of beta-carotene (10 mg/kg body wt) with a meal. Animals were killed at various time points over an 11-d period. Blood and tissue samples were extracted and analyzed for beta-carotene by HPLC. Peak serum beta-carotene levels (0.68 +/- 0.18 mumol/L) were observed 8 h after the test meal. beta-Carotene was essentially cleared from the blood by 76 h. Peak beta-carotene concentrations (nmol/g) were observed between 8 and 16 h after ingestion for liver (1.20 +/- 0.04), lung (0.042 +/- 0.012), kidney (0.090 +/- 0.015) and spleen (0.076 +/- 0.012). Ferret liver also seemed to contain a variety of other polar and nonpolar carotenoids. Ferrets were shown to absorb beta-carotene from a meal and have a consistent serum response pattern. Absorbed beta-carotene is differentially distributed in a variety of tissues. The ferret seems to be a useful model for the study of beta-carotene absorption and metabolism.

Interactions of oral beta-carotene and canthaxanthin in ferrets.

Interactive effects of an oral dose of equal quantities of beta-carotene and either canthaxanthin or lycopene on serum and tissue beta-carotene accumulations were investigated in domestic ferrets. Like humans, ferrets absorb a substantial portion of ingested beta-carotene intact and accumulate it in tissues. After the ferrets ingested a low carotenoid purified diet for 13 d, they were randomly assigned to one of two groups of six animals. One group was dosed with beta-carotene (10 mg/kg body weight) and the other with beta-carotene and either canthaxanthin (Experiment 1) or lycopene (Experiment 2) (10 mg/kg body weight for each). In Experiment 1, ferrets that received a combined dose of beta-carotene and canthaxanthin had serum beta-carotene concentrations that were significantly lower at 8, 12 and 24 h post-dosing (P < 0.05), compared with those that received an individual dose of beta-carotene; liver, adrenal and kidney beta-carotene concentrations were also significantly reduced. In Experiment 2, ferrets that received a combined dose of lycopene and beta-carotene had lower serum and tissue beta-carotene concentrations than in those that received beta-carotene alone; the differences were not statistically significant with the exception of serum beta-carotene concentrations at 24 h post-dosing. The results suggest that, at the doses given, a concurrent oral canthaxanthin dose has a specific antagonistic effect on the bioavailability of a beta-carotene dose in ferrets.

Soybean phytochemicals inhibit the growth of transplantable human prostate carcinoma and tumor angiogenesis in mice.

The objectives of our studies are to characterize the ability of dietary soybean components to inhibit the growth of prostate cancer in mice and alter tumor biomarkers associated with angiogenesis. Soy isoflavones (genistein or daidzein) or soy phytochemical concentrate inhibit the growth of prostate cancer cells LNCaP, DU 145 and PC-3 in vitro, but only at supraphysiologic concentrations, i.e., 50% inhibitory concentration (IC(50)) > 50 micromol/L. G2-M arrest and DNA fragmentation consistent with apoptosis of prostate cancer cells are also observed at concentrations causing growth inhibition. In contrast, the in vitro proliferation of vascular endothelial cells was inhibited by soy phytochemcials at much lower concentrations. We evaluated the ability of dietary soy phytochemical concentrate and soy protein isolate to inhibit the growth of the LNCaP human prostate cancer in severe combined immune-deficient mice. Mice inoculated subcutaneously with LNCaP cells (2 x 10(6)) were randomly assigned to one of the six dietary groups based on the AIN-76A formulation for 3 wk. A 2 x 3 factorial design was employed with two protein sources (20%, casein vs. soy protein) and three levels of soy phytochemical concentrate (0, 0.2 and 1.0% of the diet). Soy components did not alter body weight gain or food intake. Compared with casein-fed controls, the tumor volumes after 3 wk were reduced by 11% (P = 0.45) by soy protein, 19% (P = 0.17) by 0.2% soy phytochemical concentrate, 28% by soy protein with 0.2% soy phytochemical concentrate (P < 0.05), 30% by 1.0% soy phytochemical concentrate (P < 0.05) and 40% by soy protein with 1.0% soy phytochemical concentrate (P < 0.005). Histologic examination of tumor tissue showed that consumption of soy products significantly reduced tumor cell proliferation, increased apoptosis and reduced microvessel density. The angiogenic protein insulin-like growth factor-I was reduced in the circulation of mice fed soy protein and phytochemical concentrate. Our data suggest that dietary soy products may inhibit experimental prostate tumor growth through a combination of direct effects on tumor cells and indirect effects on tumor neovasculature.