The effect of genistein supplementation on performance and antioxidant status of Japanese Quail under heat stress

Genistein, a phytoestrogen found in soybeans, is a powerful antioxidant. We evaluated the effects of genistein supplementation on performance, carcass characteristics, levels of malondialdehyde (MDA), homocysteine, vitamins C, E, A in Japanese quail (Coturnix coturnix japonica) exposed to high ambient temperature of 34°C. Two hundred and forty Japanese quails (10 d old) were randomly assigned to eight treatment groups consisting of 10 replicates of three birds. The birds were kept in an environmental controlled room either for 24 h/d at 22°C with (thermoneutral, TN groups) or for 16 h/d at 22°C and for 8 h/d (09.00 am to 05.00 pm) at 34°C (heat stress, HS groups). Birds were fed either a basal (control) diet (TN and HS) or the basal diet supplemented with 200, 400 or 800 mg of genistein per kg of diet. Heat exposure decreased birds' performance when basal diet was fed. Increase in feed intake and body weight, and improvement of feed efficiency and carcass traits were found in genistein-supplemented quails reared under heat stress conditions. Growth rate and feed efficiency improved in quails reared under thermo-neutral conditions as well. Concentration of serum vitamins C, E, and A increased in supplemented birds reared at high temperature, while non-significant changes occurred in TN groups. With genistein supplementation homocysteine levels in serum and MDA levels in serum and liver decreased in all birds of both TN and HS groups. Effects of genistein were relatively greater in heat-stressed quails than in quails kept under thermo-neutral conditions. Results of the present study suggest that supplementation with genistein can be considered to be protective by reducing the negative effects of oxidative stress induced by heat stress in quail.     

Genistein-induced G2/M cell cycle arrest of human intestinal colon cancer Caco-2 cells is associated with Cyclin B1 and Chk2 down-regulation

Genistein is an isoflavonic phyto-oestrogen contained in soya beans. It is thought to display anti-cancer effects. This study was designed to investigate its effect on human intestinal colon cancer Caco-2 cells. MTT assay, flow cytometric analysis and western blotting were used to investigate the effect of genistein on cell proliferation, cell cycle progression and protein alterations of selected cell cycle-related proteins in Caco-2 cells. Our results showed that genistein and daidzein significantly suppressed cell proliferation. Genistein treatment was demonstrated to modulate cell cycle distribution through accumulation of cells at G2/M phase, with a significant decreasing effect of Cyclin B1 and Serine/threonine-protein kinase 2 (Chk2) proteins expression. However, daidzein did not alter the cell cycle progression in Caco-2 cells. All these observation strongly indicate that genistein has anti-proliferative effect in human intestinal colon cancer Caco-2 cells through the down-regulation of cell cycle check point proteins, Cyclin B1 and Chk2.     

Enhanced extraction genistein from pigeon pea [Cajanus cajan (L.) Millsp.] roots with the biotransformation of immobilized edible Aspergillus oryzae and Monacus anka and antioxidant activity evaluation

A new method of enhanced extraction genistein from pigeon pea [Cajanus cajan (L.) Millsp.] roots with the biotransformation of immobilized edible Aspergillus oryzae and Monacus anka, was investigated. It showed that immobilized Aspergillus oryzae and Monacus anka on sodium alginate effectively supported the highest genistein extraction yield by screening microorganism tests. After biotransformation process with immobilized Aspergillus oryzae and Monacus anka under 30 °C, pH 6.0, 2 days, liquid-solid ratio 12: 1 (mL/g), the extraction yield of genistein reached 1.877 mg/g, which was 2.65-fold to that of normal extraction yield. Moreover, IC₅₀ values of the extracts measured by DPPH-radical scavenging test and β-Carotene-linoleic acid bleaching test were 0.737 mg/mL and 0.173 mg/mL (control sample 1.117 mg/mL and 0.216 mg/mL), respectively. SOD (Super Oxygen Dehydrogenises) activity of the extracts treated with immobilized microorganism which was stronger than that of the untreated pigon pea roots (1.44 U/mg) at the concentration of protein (0.9375 μg/mL) was 1.83 U/mg. The developed method could be an alternative method for the enhanced extraction of genistein from plants and could be potentially applied in the food industry     

Synergistic chemoprotective mechanisms of dietary phytoestrogens in a select combination against prostate cancer

Combination of dietary phytoestrogens with diverse molecular mechanisms may enhance their anticancer efficacy at physiological concentrations, as evidenced in epidemiological studies. A select combination of three dietary phytoestrogens containing 8.33 μM each of genistein (G), quercetin (Q) and biochanin A (B) was found to be more potent in inhibiting the growth of androgen-responsive prostate cancer cells (LNCaP) as well as DU-145 and PC-3 prostate cancer cells in vitro than either 25 μM of G, B or Q or 12.5+12.5 μM of G+Q, Q+B or G+B. Subsequent mechanistic studies in PC-3 cells indicated that the action of phytoestrogens was mediated both through estrogen receptor (ER)-dependent and ER-independent pathways as potent estrogen antagonist ICI-182780 (ICI, 5 μM) could not completely mask the synergistic anticancer effects, which were sustained appreciably in presence of ICI. G+Q+B combination was significantly more effective than individual compounds or their double combinations in increasing ER-β, bax (mRNA expression); phospho-JNK, bax (protein levels); and in decreasing bcl-2, cyclin E, c-myc (mRNA expression); phospho-AKT, phospho-ERK, bcl-2, proliferating cell nuclear antigen (protein levels) in PC-3 cells. Phytoestrogens also synergistically stimulated caspase-3 activity. Our findings suggest that selectively combining anticancer phytoestrogens could significantly increase the efficacy of individual components resulting in improved efficacy at physiologically achievable concentrations. The combination mechanism of multiple anticancer phytochemicals may be indicative of the potential of some vegetarian diet components to elicit chemopreventive effects against prostate cancer at their physiologically achievable concentrations, in vivo.     

Genistein-induced mitotic arrest of gastric cancer cells by downregulating KIF20A, a proteomics study

Genistein exerts its anticarcinogenic effects by inducing G2/M arrest and apoptosis of cancer cells. However, the precise molecular mechanism of action of genistein has not been completely elucidated. In this study, we used quantitative proteomics to identify the genistein-induced protein alterations in gastric cancer cells and investigate the molecular mechanism responsible for the anti-cancer actions of genistein. Total 86 proteins were identified to be regulated by genistein, most of which were clustered into the regulation of cell division and G2/M transition, consistent with the anti-cancer effect of genistein. Many proteins including kinesin family proteins, TPX2, CDCA8, and CIT were identified for the first time to be regulated by genistein. Interestingly, five kinesin family proteins including KIF11, KIF20A, KIF22, KIF23, and CENPF were found to be simultaneously downregulated by genistein. Significantly decreased KIF20A was selected for further functional studies. The silencing of KIF20A inhibited cell viability and induced G2/M arrest, similar to the effects of genistein treatment in gastric cancer. And the silencing of KIF20A also increased cancer cell sensitivity to genistein inhibition, whereas overexpression of KIF20A markedly attenuated genistein-induced cell viability inhibition and G2/M arrest. These observations suggested that KIF20A played an important role in anti-cancer actions of genistein, and thus may be a potential molecular target for drug intervention of gastric cancer.     

Genistein induces topoisomerase IIbeta- and proteasome-mediated DNA sequence rearrangements: Implications in infant leukemia

Genistein is a bioflavonoid enriched in soy products. However, high levels of maternal soy consumption have been linked to the development of infant leukemia ALL and AML. The majority of infant leukemia is linked to mixed lineage leukemia gene (MLL) translocations. Previous studies have implicated topoisomerase II (Top2) in genistein-induced infant leukemia. In order to understand the roles of the two Top2 isozymes in and the molecular mechanism for genistein-induced infant leukemia, we carried out studies in vitro using purified recombinant human Top2 isozymes, as well as studies in cultured mouse myeloid progenitor cells (32Dc13) and Top2β knockout mouse embryonic fibroblasts (MEFs). First, we showed that genistein efficiently induced both Top2α and Top2β cleavage complexes in the purified system as well as in cultured mouse cells. Second, genistein induced proteasomal degradation of Top2β in 32Dc13 cells. Third, the genistein-induced DNA double-strand break (DSB) signal, γ-H2AX, was dependent on the Top2β isozyme and proteasome activity. Fourth, the requirement for Top2β and proteasome activity was mirrored in genistein-induced DNA sequence rearrangements, as monitored by a DNA integration assay. Together, our results suggest a model in which genistein-induced Top2β cleavage complexes are processed by proteasome, leading to the exposure of otherwise Top2β-concealed DSBs and subsequent chromosome rearrangements, and implicate a major role of Top2β and proteasome in genistein-induced infant leukemia.     

金雀异黄素合成品诱导人胃癌细胞SGC-7901凋亡的体外研究

目的:探讨金雀异黄素合成品诱导人胃癌细胞SGC-7901凋亡的作用机制。方法:采用2.5mg·L~(-1)、5.0mg·L~(-1)、10.0mg·L~(-1)和20.0mg·L~(-1)的金雀异黄素处理人胃癌细胞SGC-7901后,用流式细胞仪检测细胞凋亡率,电镜下观察细胞形态变化,RT-PCR法检测凋亡相关基因表达。结果:10.0mg·L~(-1),20.0mg·L~(-1)金雀异黄素能诱导胃癌细胞SGC-7901凋亡,凋亡率与剂量正相关(相关系数r=0.9830),10.0mg·L~(-1)金雀异黄素诱导胃癌细胞SGC-7901发生凋亡的形态学改变,2.5mg·L~(-1)、5.0mg·L~(-1)、10.0mg·L~(-1)和20.0mg·L~(-1)金雀异黄素使Bcl-2mRNA表达下调,Fas mRNA表达上调。结论:金雀异黄素能诱导胃癌细胞SGC-7901凋亡,降低Bcl-2 mRNA表达,增加Fas mRNA表达为其诱发SGC-7901细胞凋亡的机制之一。     

Biotransformation of genistein in the rat: elucidation of metabolite structure by product ion mass fragmentology

Biotransformation of the phytoestrogen [¹⁴C]genistein was investigated in male and female rats by application of narrow-bore radio-HPLC-MSⁿ (LCQ, Finnigan) to determine intermediates in metabolism. Urine contained five metabolites, Gm1–Gm5, 24 h after dosing by gavage with [¹⁴C]genistein (4 mg kg⁻¹). Structural analysis following ESI revealed molecular ions [M+H]⁺ of m/z 447, 449, 273, and 271 for metabolites Gm2, Gm3, Gm5 and genistein, respectively and an [M–H]⁻ of m/z 349 for Gm4. Metabolite structure was deduced by evaluation of product ion spectra derived from unlabelled and [¹⁴C]-labelled ions and sensitivity to treatment with β-glucuronidase. These studies indicated identity of metabolites with genistein glucuronide (Gm2), dihydrogenistein glucuronide (Gm3), genistein sulphate (Gm4) and dihydrogenistein (Gm5). Detection of the β-glucuronidase resistant major metabolite Gm1 by ESI was poor and so was analysed by negative ion APCI; this revealed a deprotonated molecular ion of m/z 165 which had chromatographic and mass spectral properties consistent with authentic 4-hydroxyphenyl-2-propionic acid, a novel metabolite of genistein. In vitro metabolism studies with anaerobic caecal cultures derived from male and female rats revealed metabolism of genistein to Gm1 via Gm5 and an additional metabolite (Gm6) which was identified from product ion spectra as 6′-hydroxy-O-desmethylangolensin. Biotransformation of genistein by both isolated hepatocytes and precision-cut liver slices was limited to glucuronidation of parent compound. Commonality of genistein metabolites found in rats with those reported in man suggest similar pathways of biotransformation, primarily involving gut micro-flora.