Jimmy D. Browning

Phytoestrogens in Common Herbs Regulate Prostate Cancer Cell Growth in Vitro

Abstract: Prostate cancer is an important public health problem in the United States. Seven phytoestrogens found in common herbal products were screened for estrogen receptor binding and growth inhibition of androgen-insensitive (PC-3) and androgen-sensitive (LNCaP) human prostate tumor cells. In a competitive 3H-estradiol ligand binding assay using mouse uterine cytosol, 2.5 μM quercetin, baicalein, genistein, epigallocatechin gallate (EGCG), and curcumin displaced > 85% of estradiol binding, whereas apigenin and resveratrol displaced > 40%. From growth inhibition studies in LNCaP cells, apigenin and curcumin were the most potent inhibitors of cell growth, and EGCG and baicalein were the least potent. In PC-3 cells, curcumin was the most potent inhibitor of cell growth, and EGCG was the least potent. In both cell lines, significant arrest of the cell cycle in S phase was induced by resveratrol and EGCG and in G2M phase by quercetin, baicalein, apigenin, genistein, and curcumin. Induction of apoptosis was induced by all of the 7 compounds in the 2 cell lines as shown by TUNEL and DNA fragmentation assays. Androgen responsiveness of the cell lines did not correlate with cellular response to the phytoestrogens. In conclusion, these 7 phytoestrogens, through different mechanisms, are effective inhibitors of prostate tumor cell growth.

Sorghum 3-Deoxyanthocyanins Possess Strong Phase II Enzyme Inducer Activity and Cancer Cell Growth Inhibition Properties

3-Deoxyanthoxyanins (3-DXA) possess unique chemical and biochemical properties and may be useful in helping reduce incidence of gastrointestinal cancer. This study tested sorghum extracts rich in 3-DXA as well as isolated and synthetic 3-DXA for potential to induce activity of phase II enzymes in murine hepatoma cells using the NAD(P)H:quinone oxidoreductase (NQO) assay and to inhibit proliferation of the HT-29 human colon cancer cells using MTT and PicoGreen assays. Crude black sorghum extract that contained high levels of methoxylated 3-DXA was a strong inducer of NQO activity (3.0 times at 50 microg/mL), compared to red or white sorghum extracts with low or no methoxylated 3-DXA (1.6 times at 200 microg/mL). All sorghum extracts had strong antiproliferative activity against HT-29 cells after 48 h of incubation (IC(50) = 180-557 microg/mL). Among isolated fractions, nonmethoxylated 3-DXA were very effective against HT-29 cell growth (IC(50) = 44-68 microM at 48 h), but were noninducers of NQO. On the other hand, the methoxylated 3-DXA had both strong antiproliferative activity (IC(50) < 1.5-53 microM) and NQO inducer activity (2-3.7 times). Dimethoxylated 3-DXA were more potent than monomethoxylated analogues. Methoxylation of 3-DXA is essential for NQO activity and also enhances tumor cell growth inhibition.

Aggressive Prostate Cancer Is Prevented in ERαKO Mice and Stimulated in ERβKO TRAMP Mice

Previous evidence suggests soy genistein may be protective against prostate cancer, but whether this protection involves an estrogen receptor (ER)-dependent mechanism is unknown. To test the hypothesis that phytoestrogens may act through ERα or ERβ to play a protective role against prostate cancer, we bred transgenic mice lacking functional ERα or ERβ with transgenic adenocarcinoma of mouse prostate (TRAMP) mice. Dietary genistein reduced the incidence of cancer in ER wild-type (WT)/transgenic adenocarcinoma of mouse prostate mice but not in ERα knockout (KO) or ERβKO mice. Cancer incidence was 70% in ERWT mice fed the control diet compared with 47% in ERWT mice fed low-dose genistein (300 mg/kg) and 32% on the high-dose genistein (750 mg/kg). Surprisingly, genistein only affected the well differentiated carcinoma (WDC) incidence but had no effect on poorly differentiated carcinoma (PDC). No dietary effects have been observed in either of the ERKO animals. We observed a very strong genotypic influence on PDC incidence, a protective effect in ERαKO (only 5% developed PDC), compared with 19% in the ERWT, and an increase in the incidence of PDC in ERβKO mice to 41%. Interestingly, immunohistochemical analysis showed ERα expression changing from nonnuclear in WDC to nuclear in PDC, with little change in ERβ location or expression. In conclusion, genistein is able to inhibit WDC in the presence of both ERs, but the effect of estrogen signaling on PDC is dominant over any dietary treatment, suggesting that improved differential targeting of ERα vs. ERβ would result in prevention of advanced prostate cancer.

Neuropeptide Y fails to normalize food intake in zinc-deficient rats.

Zinc deprivation results in decreased and cyclic food intake in rats. We determined the response of zinc-deprived rats to neuropeptide Y (NPY). In a preliminary experiment, rats were fed a low (-Zn; <1 mg/kg) or adequate zinc diet (+Zn; 100 mg/kg) for 4 days. NPY (5 or 10μg) was then administered via an intracerebroventricular (ICV) cannula and food intake measured for 4h. NPY stimulated food intake in all rats, but the difference in food intake due to zinc deprivation persisted. In a subsequent experiment, rats were fed the low zinc and adequate zinc diets for 4, 5 or 6 days. Food intake was suppressed in rats fed the low zinc compared to the adequate zinc diet on all of these days. When NPY (10 μg) was administered at the onset of the light cycle, the food intake was approximately 2.5-fold greater regardless of dietary zinc status, but the amount of food consumed by rats fed low zinc was approximately one-half the quantity consumed by NPY-stimulated zinc-adequate rats. NPY administered at the onset of dark failed to stimulate food intake in either dietary group although the total intake difference due to zinc status persisted. ICV administration of 5nmol of zinc prior to NPY injection failed to correct the food intake response of the zinc-deficient rats. We conclude that the basis of the reduced food intake of zinc-deficient rats does not relate to NPY quantity or release, or to impairment of its signal transduction. There appears to be another undefined factor that limits food intake in zinc deficiency.

In vitro addition of glutathione to blood from zinc-deficient rats corrects platelet defects: Impaired aggregation and calcium uptake

Abstract Zinc deficiency in rats impairs platelet aggregation, and this defect is associated with the failure of platelets to take up external calcium. Recent data show that the red cell plasma membrane from zinc-deficient rats contains a lower than normal concentration of protein sulfhydryl groups, a defect that can be corrected readily in vivo by zinc repletion. The purpose of this study was to determine the effect of zinc deficiency on sulfhydryl concentration of platelet membrane proteins and to determine if impaired platelet function can be corrected in vitro by treatment of blood with glutathione (GSH), a physiological reducing agent. Immature male rats were fed low zinc diets based on egg white or EDTA-treated soy protein (

Zinc Deficiency and Peripheral Neuropathy in Chicks

Abstract Zinc-deficient chicks develop an arthritic-like neuromuscular disorder. They walk with a stilted gait and tend to remain in a squat position, bearing little weight on the legs. The purpose of this study was to determine the basis of the syndrome by making electrophysiologic measurements of nerve function. Chicks were fed low zinc (6 mg/kg) and zinc-adequate (50 mg/kg) diets, the latter ad libitum and pair-fed. At the end of 3 weeks, sciatic nerve function was determined in vivo by use of an electrodiagnostic system. Motor nerve conduction velocity was significantly lower in chicks fed the low zinc than in those fed the zinc-adequate diet. Zinc repletion of the 2-week depleted chicks was achieved by feeding the adequate diet for 2 weeks. Repletion for this period cured clinical signs and restored nerve conduction velocity to normal, but reversal did not occur within 1 week. It was concluded that the abnormal posture and locomotion of zinc deficiency are associated with peripheral neuropathy.

Effect of zinc deficiency on enzyme activities in rat and pig erythrocyte membranes.

Abstract There is need for a reliable index of zinc status in humans. Considering the importance of zinc in membrane function, activities of erythrocyte membrane enzymes have been measured in animals of low and normal zinc status as possible indices. Immature rats and neonatal pigs were fed low and adequate zinc diets; the latter was fed both ad libitum and restricted so as to control for food intake effects. Low rates of gain and plasma zinc concentrations demonstrated that animals fed the low zinc diets were of low zinc status. Erythrocyte membranes were prepared and assayed for Na,K-ATPase, 5′-nucleotidase, and calcium-ATPase activities. Na,K-ATPase activity was not affected by zinc status, but 5′-nucleotidase was significantly lower in deficient animals of both species than in controls, whose food intake was restricted to maintain comparable weight (2.76 vs 3.94 nmol/hr/mg of protein in rats and 60.5 vs 119 in pigs). The basal calcium-ATPase activities were also decreased by low zinc status in both species. Addition of calmodulin in vitro stimulated activity two-fold to four-fold and resulted in the same maximal activities for all treatments. The results show that erythrocyte membrane 5′-nucleotidase activity is an index of zinc status in these species. It is suggested that the decreased membrane calcium-ATPase activity in zinc deficiency is caused by a defect in calmodulin metabolism.

Chelation of Extracellular Zinc Inhibits Proliferation in 3T3 Cells Independent of Insulin-Like Growth Factor-I Receptor Expression

Abstract Depletion of zinc inhibits growth in animals and proliferation of cultured cells. Additionally, zinc can serve as an antioxidant protecting many compounds, including proteins, from oxidation. Regulation of cell division also involves insulinlike growth factor type I (IGF-I) and its receptor, especially during late G1 phase, allowing progression of the cell to S phase with subsequent DNA synthesis. We examined the effects of zinc depletion from the culture media of Swiss 3T3 cells on the cell cycle and IGF-I receptor expression. Cells were exposed to reduced fetal bovine serum concentrations to induce growth arrest, then returned to normal fetal bovine serum concentrations with the divalent cation chelator diethylenetriamine pentaacetic acid. Reducing the fetal bovine serum concentration did not induce quiescence in the cells as previously suggested. Zinc depletion reduced the proliferative fraction (S and G2/M phases) of the cell cycle. The addition of glutathione to the zinc-depleted media partially returned the proliferative fraction to the control level. Fetal bovine serum deprivation reduced IGF-I receptor expression whereas the absence of zinc had little effect on receptor expression. We conclude that depletion of zinc from culture media inhibits 3T3 cell proliferation independent of insulin-like growth factor-I receptor expression, and part of this inhibition is due to the antioxidant capacity of this divalent cation.

Effect of zinc deficiency and food restriction in the pig on erythrocyte fragility and plasma membrane composition

Abstract Neonatal pigs (3 groups of 6) were fed for 28 days one of three diets, a low zinc diet (4.6 mg/kg) ad libitum (−ZnAL), an adequate zinc diet (103 mg/kg) ad libitum (+ZnAL) or the latter diet restricted (+ZnRF) to allow gain comparable to those fed −ZnAL. Osmotic fragility was measured, and erythrocyte membranes were isolated and analyzed for zinc, phospholipid fatty acids, and SDS-PAGE protein profiles. The −ZnAL diet produced severe zinc deficiency that resulted in increased osmotic fragility and decreased membrane zinc concentration, compared to +ZnAL. The linoleic acid contents of both phosphatidylcholine and phosphatidyl-ethanolamine were decreased while that of oleic acid was increased. The cholesterol/phospholipid ratio was higher than that of the +ZnAL group. of the membrane proteins, band 3 was lower while ankyrin was higher than those of the +ZnAL group. Relative to the abnormal fragility, decreased membrane zinc is considered most significant.

Low zinc status impairs calcium uptake by hippocampal synaptosomes stimulated by potassium but not by N-methyl-D-aspartate

Zinc deficiency results in neuropathology affecting both the peripheral and central nervous systems. A previous study showed that decreased calcium uptake by cortical synaptosomes was associated with the peripheral neuropathy in guinea pigs. Deficiency impaired the calcium uptake stimulated by high potassium and by additional glutamate. In this study, the effect of zinc status on potassium-stimulated and agonist (glutamate and N-methyl-D-aspartate [NMDA]-stimulated calcium uptake by both cortical and hippocampal synaptosomes was examined. Groups of guinea pigs were allowed to consume a low zinc (<1 mg/kg) diet ad libitum (−ZN) and an adequate zinc (100 mg/kg) diet either ad libitum (+ AL) or restricted (+RF). Synaptosomes were prepared from cortex and hippocampus and calcium uptake measured using 45Ca. Potassium-stimulated calcium uptake by cortical and hippocampal synaptosomes was significantly lower in synaptosomes from zinc deficient guinea pigs than in controls, 15% and 20%, respectively. Glutamate-stimulated calcium uptake by cortical synaptosomes from deficient animals was 32% less than that of controls; there was a similar trend in hippocampal synaptosomes. Zinc deficiency had no effect on the NMDA-stimulated uptake by synaptosomes from either source. Impairment of voltage-gated calcium channels appears to account for the decreased calcium uptake and may explain the neuropathology observed in zinc deficiency.