Long-term effects of dietary isoflavones on uterine gene expression profiles

Isoflavones (ISOs) are bioactive food ingredients of the traditional East Asian diet and currently discussed as alternatives to classical hormone replacement therapies and for reducing the prevalence of hormone-dependent cancers. Although there are many studies on ISOs, not much is known about their long-term effects.Therefore, we performed an animal experiment analyzing the effects of three different diets: a phytoestrogen-free diet, a diet supplemented with genistein (700 μg/g diet) and an ISO-high diet (232 μg daidzein and 240 μg genistein/g) at two distinct time points, juvenile (21 days) and adult (97 days). Exposure started prior to mating of the parents and throughout the life of the offspring.We observed a stronger increase of uterine wet weights in juvenile offspring with genistein exposure (1018 ± 350 mg/kg BW) than with ISO-high diet (497 ± 133 mg/kg BW). Whereas the expression of proliferation related genes (PCNA; Ki67; IGF-1; IGF-1R), analyzed by real-time-qPCR and Western blot, were significantly down-regulated in juvenile animals exposed to genistein. Additionally, genistein exposure led to estrogenic responses, observed upon increase of complement C3 and decrease of estrogen receptors gene expressions, while the exposure to ISO-high diet did not show these effects.In conclusion, both the time point on which phytoestrogen exposure starts together with the composition of the ingested phytoestrogen containing diet are of great importance for the biological response of the offspring.     

Genistein induces apoptosis by stabilizing intracellular p53 protein through an APE1-mediated pathway

Genistein (GEN) has been previously shown to have a proapoptotic effect on cancer cells through a p53-dependent pathway, the mechanism of which remains unclear. One of its intracellular targets, APE1, protects against apoptosis under genotoxic stress and interacts with p53. In this current study, we explored the mechanism of the proapoptotic effect of GEN by examining the APE1–p53 protein–protein interaction. We initially showed that the p53 protein level was elevated in GEN-treated human non-small lung cancer A549 cells and cervical cancer HeLa cells. By examining both protein synthesis and degradation, we found that GEN enhances p53 intracellular stability by interfering with the interaction of APE1 and p53, which provided a plausible explanation for how GEN initiates apoptosis. Furthermore, we found that the interaction between APE1 and p53 is important for the degradation of p53 and is dependent on the redox domain of APE1 by utilizing the redox domain mutant APE1 C65A. Our data suggest that the degradation of wild-type p53 is blocked when the redox domain of APE1 is masked or interrupted. Based on this evidence, we hereby report a novel mechanism of p53 degradation through an APE1-mediated, redox-dependent pathway.     

Dual effects of isoflavonoids from Pueraria lobata roots on estrogenic activity and anti-proliferation of MCF-7 human breast carcinoma cells

Pueraria lobata root (PLR), well known as Kudzu root, has recently become commercially available in Western dietary supplements for menopausal symptoms. The scientific basis for its action has been attributed to the action of phytoestrogens. This study aimed to investigate the estrogen-like activity of isoflavonoids isolated from P. lobata root and their safety with respect to their effect on breast cancer cell proliferation. In an E-screen assay, crude MeOH extract of PLR significantly increased the proliferation of MCF-7 cells in a concentration-dependent manner. Among the four fractions obtained by solvent fractionation of MeOH extract, the n-BuOH fraction had significant estrogen-like activities at all concentrations tested. Phytochemical analysis of the n-BuOH fraction led to the isolation of 10 isoflavones (1–10), among which genistein (10) had significant estrogen-like activities at all concentrations tested. These activities were significantly enhanced by treatment with genistein and 17β-estradiol compared with 17β-estradiol alone, and this effect was mediated by decreased expression of estrogen receptor (ER)α and phospho-ERα in MCF-7 cells. In a cell cytotoxicity assay, genistein (10) exhibited significant cytotoxicity in both ER-positive MCF-7 and ER-negative MDA-MB-231 breast cancer cells. This cytotoxicity was characterized by the induction of apoptotic cells stained with annexin V conjugated with Alexa Fluor 488 and involved activation of mitochondria-independent and -dependent apoptosis pathways in MCF-7 cells. Our results demonstrated that genistein (10) has estrogen-like effects dependent on ER pathway activation and anti-proliferative effects mediated by the apoptosis pathway rather than the ER pathway in MCF-7 breast cancer cells.     

Pharmacological studies of the large-scaled purified genistein from Huaijiao (Sophora japonica – Leguminosae) on anti-osteoporosis

In this report, we used genistein that was extracted from a Chinese herbal medicine Huaijiao (Sophora japonica – Leguminosae) to evulate its pharmacological function on anti-osteoporosis. This genistein is purified in a large-scale production from Huaijiao by a state-of-art method as described by Tian et al. [2004. The preparation of genistein and LC-MS/MS on-line analysis. Drug Devel. Res. 61, 6–12]. Chemical structure of the isolated genistein was examined by using various techniques including nuclear magnetic resonant spectrum, infrared absorption spectrum, ultraviolet absorption spectrum and mass spectrum, and was proved to be identical to those purified from soybean in a small scale as previously reported. We randomly divided female SD rats into 6 groups, including control, ovariectomized model, Nilestriol-treated, and three level of dosages of genistein-treated. We evaluated the pharmacological effects of genistein against osteoporosis by measuring the bone density of femur and bone mineral group including calcium, phosphorous, and magnesium. The consequences of genistein treatment on bone histology and morphology were also determined by measuring the trabcular area, thickness and number. Our results indicated that treatment with a 4.5 or 9 mg/kg dosage of genistein could also prevent osteoporosis significantly at the 4th week after treatment. In comparison with the anti-osteoporosis effects of soybean genistein, the genistein extracted from Huaijiao has the same beneficial effect on anti-osteoporosis.     

Efectos del uso del 17 β-estradiol y la genisteína en la enfermedad de Alzheimer en mujeres con menopausia

The use of 17 β-estradiol and genistein in women with menopause helps in the reduction of vasomotor symptoms and cognitive improvement. There is evidence on the use of certain flavonoids such as genistein, which has a potentially neuroprotective role in neurodegenerative diseases such as Alzheimer's. Scientific evidence on the effects of phytoestrogens and genistein during menopause and their effect on cognition are scarce, however, in the present review it was found that the intervention with 17 β-estradiol has positive effects on cognition in women with Alzheimer's disease. In addition, the use of genistein, daidzein or any supplement based on isoflavones may influence vasomotor symptoms. 17 β-estradiol supplements in women in early menopause and with some degree of cognitive impairment may have beneficial effects.     

蛋白酪氨酸激酶抑制剂Genistein抑制肺癌细胞A549体外侵袭作用的研究

观察蛋白酪氨酸激酶抑制剂Genistein对人肺腺癌细胞株A549细胞侵袭能力的影响,探讨Genistein抑制肺癌细胞侵袭的可能机制。以不同浓度Genistein（20μmol/L和40μmol/L）作用于A549细胞3 d后,分别用基质胶侵袭模型、黏附基质分析、Transwell小室趋化运动模型、细胞骨架蛋白染色及RT-PCR法来研究药物处理后细胞侵袭、黏附、运动、聚合型骨架蛋白（F-actin）以及基质金属蛋白酶基因表达的改变。经Genistein处理后,A549细胞的F-actin聚合减少,侵袭能力明显下降,趋化运动能力降低,基质金属蛋白酶抑制剂（TIMP-1）基因相对表达量增加,但黏附率没有降低。Genistein可降低肺癌细胞的迁移、侵袭能力。F-actin聚合减少,TIMP-1的相对表达量增加,可能是Genistein抑制肺癌细胞侵袭的机制之一。     

Genistein induces G2/M cell cycle arrest and apoptosis of human ovarian cancer cells via activation of DNA damage checkpoint pathways

Genistein is a major isoflavonoid in dietary soybean, commonly consumed in Asia. Genistein exerts inhibitory effects on the proliferation of various cancer cells and plays an important role in cancer prevention. However, the molecular and cellular mechanisms of genistein on human ovarian cancer cells are still little known. We show that exposure of human ovarian cancer HO-8910 cells to genistein induces DNA damage, and triggers G2/M phase arrest and apoptosis. Furthermore, we also found that checkpoint proteins ATM and ATR are phosphorylated and activated in the cells treated with genistein. It is also shown that genistein increases the phosphorylation and activation of Chk1 and Chk2, which results in the phosphorylation and inactivation of phosphatases Cdc25C and Cdc25A, and thereby the phosphorylation and inactivation of Cdc2 which arrests cells in G2/M phase. Moreover, genistein enhances the phosphorylation and activation of p53, while decreases the ratio of Bcl-2/Bax and Bcl-xL/Bax and the level of phosphorylated Akt, which result in cells undergoing apoptosis. These results demonstrate that genistein-activated ATM-Chk2-Cdc25 and ATR-Chk1-Cdc25 DNA damage checkpoint pathways can arrest ovarian cancer cells in G2/M phase, and induce apoptosis while the cellular DNA damage is too serious to be repaired. Thus, the antiproliferative, DNA damage-inducing and pro-apoptotic activities of genistein are probably responsible for its genotoxic effects on human ovarian cancer HO-8910 cells.     

Exploring in silico affinity of flavonoids and tannins to human fibroblast growth factorinducible14 (Fn14), a member of TNF receptor super family

The Fn14 and TWEAK are the receptor and ligand respectively and their mutual recognition and binding was reported to induce pathogenesis of cancer and chronic autoimmune diseases. We had identified Fn14 as a novel target of low linear energy transfer (LET) ionizing radiation in mice population. In the present study we generated the novel homology model of human Fn14, optimized its energy and validated for authenticity by checking Ramachandran plot and also by calculating the RMSD. Based on our earlier findings with Hippophae rhamnoides, a group of flavonoids and tannins were screened for their docking potential with Fn14 at the site where its natural ligand TWEAK was binding. The comparative docking analysis showed that the order of docking, from best to least, was Genistein, Rutin, Gallic acid ethyl ester and Quercetin, respectively. The findings predicted the radiomodifying action of flavonoids and tannins. The study has immediate applications in development of non-toxic drugs/ nutraceuticals that may protect human population from harmful effects of radiation in various situations, such as nuclear accidents, occupational exposure, diagnosis or radiotherapy.