In utero and postnatal exposure to isoflavones results in a reduced responsivity of the mammary gland towards estradiol

Scope Exposure scenarios during different stages of development of an organism are discussed to trigger adverse and beneficial effects of isoflavones (ISO). The aim of this study was to investigate how in utero and postnatal ISO exposure modulates the estrogen sensitivity of the mammary gland and to identify the underlying molecular mechanisms. Methods and results Therefore, rats were exposed to either ISO‐free (IDD), ISO‐rich (IRD) or genistein‐rich diet (GRD), up to young adulthood. Proliferative activity (PCNA expression) in the mammary gland at different ages and the estrogen sensitivity of the mammary gland to estradiol (E₂) or genistein (GEN) in adult ovariectomized animals was determined and compared with different treatments. Treatment with E₂ resulted in a significant lower proliferative and estrogenic response of the mammary gland in IRD and GRD compared with IDD. This correlates to a change in the gene expression pattern and a decrease in the ratio of estrogen receptor alpha (ERα) beta (ERβ Conclusions Our results provide evidence that in utero and postnatal exposure to a diet rich in ISO but also to GEN reduces the sensitivity of the mammary gland toward estrogens and support the hypothesis that in utero and postnatal ISO exposure reduces the risk to develop breast cancer.     

Time-course regulation of quercetin on cell survival/proliferation pathways in human hepatoma cells

Quercetin, a dietary flavonoid, has been shown to possess anticarcinogenic properties, but the precise molecular mechanisms of action are not thoroughly elucidated. This study was aimed at investigating the time-course regulation effect of quercetin on survival/proliferation pathways in a human hepatoma cell line (HepG2). Quercetin induced a significant time-dependent inactivation of the major survival signaling proteins, i. e., phosphatidylinositol 3-kinase (PI 3-kinase)/protein kinase B (AKT), extracellular regulated kinase (ERK), protein kinase C-alpha (PKC-alpha), in concert with a time-dependent activation of key death-related signals: c-jun amino-terminal kinase (JNK) and PKC-delta. These data suggest that quercetin exerts a tight regulation of survival/proliferation pathways that requires the integration of different signals and persists over time, being the balance of these regulatory signals what determines the fate of HepG2 cells.     

Quercetin‐induced apoptotic cascade in cancer cells: Antioxidant versus estrogen receptor α‐dependent mechanisms

The flavonol quercetin, especially abundant in apple, wine, and onions, is reported to have anti‐proliferative effects in many cancer cell lines. Antioxidant or pro‐oxidant activities and kinase inhibition have been proposed as molecular mechanisms for these effects. In addition, an estrogenic activity has been observed but, at the present, it is poorly understood whether this latter activity plays a role in the quercetin‐induced anti‐proliferative effects. Here, we studied the molecular mechanisms of quercetin committed to the generation of an apoptotic cascade in cancer cells devoid or containing transfected estrogen receptor α (ERα; i.e., human cervix epitheloid carcinoma HeLa cells). Although none of tested quercetin concentrations increase reactive oxygen species (ROS) generation in HeLa cells, quercetin stimulation prevents the H₂O₂‐induced ROS production both in the presence and in the absence of ERα. However, this flavonoid induces the activation of p38/MAPK, leading to the pro‐apoptotic caspase‐3 activation and to the poly(ADP‐ribose) polymerase cleavage only in the presence of ERα. Notably, no down‐regulation of survival kinases (i.e., AKT and ERK) was reported. Taken together, these findings suggest that quercetin results in HeLa cell death through an ERα‐dependent mechanism involving caspase‐ and p38 kinase activation. These findings indicate new potential chemopreventive actions of flavonoids on cancer growth.     

Metabolism of quercetin and rutin by the pig caecal microflora prepared by freeze-preservation

Several studies confirm a protection of cardiovascular diseases and certain forms of cancer by dietary flavonoid intake. The bioavailability of flavonoids is influenced by the metabolism of the microflora in the intestine. Using a new in vitro model system the deglycosylation of the flavonol rutin and the degradation of its aglycone quercetin were investigated by using fresh pig caecal inocula in comparison to inocula prepared before by freeze-preservation between 6 wk and 5 months. The incubation experiments led to the same pattern of phenolic degradation products in comparable amounts in both preparations using HPLC-DAD and GC-flame ionization detection (GC-FID) or GC-MS detection within 24-48 h of incubation. With the preservation of the microbial vitality and the metabolic efficiency by freeze-preparation over several months the experimental design of microbial metabolism studies will be independent in time and locality.     

Mammary epithelial proliferation and estrogen receptor alpha expression in prepubertal heifers: effects of ovariectomy and growth hormone

The objectives of this study were to determine the effects of ovariectomy and growth hormone on mammary epithelial cell proliferation and estrogen receptor alpha (ER alpha) expression within the bovine mammary gland. Two experiments were performed. In the first experiment, eight Holstein heifer calves aged between 1 and 3 mo were ovariectomized, while six calves served as controls. At 6 mo of age, calves were treated with bromodeoxyuridine (BrdU) to label proliferating cells and sacrificed 2 h later. Coinciding with reduced mammary mass (304 +/- 25 vs. 130 +/- 21 g), proliferation of mammary epithelial cells was significantly lower in ovariectomized heifers compared to control heifers (2.24 vs. 0.25%). ER alpha expression was restricted to mammary epithelial cells and was not observed within intra-lobular stroma of parenchymal tissue. The proportion of ER alpha positive cells was significantly higher in ovariectomized heifers than in controls (36.1% +/- 2.2 vs. 46.7% +/- 2.4). In the second experiment, mammary biopsies were taken from five 6-mo-old heifers, immediately preceding and 7 d following a single injection of bovine growth hormone. Mammary epithelial cell proliferation (assessed by incorporation of 3H-thymidine) was increased by growth hormone. The proportion of ER alpha positive mammary epithelial cells was not increased by growth hormone. In conclusion, reduced mammary epithelial cell proliferation following ovariectomy was associated with an increase in ER alpha expression, whereas increased proliferation caused by bovine growth hormone was not associated with changes in the proportion of ER alpha positive cells.     

Enterolactone restricts the proliferation of the LNCaP human prostate cancer cell line in vitro

Ecological data suggest a long‐term diet high in plant material rich in biologically active compounds, such as the lignans, can significantly influence the development of prostate cancer over the lifetime of an individual. The capacity of a pure mammalian lignan, enterolactone (ENL), to influence the proliferation of the LNCaP human prostate cancer cell line was investigated as a function of cell density, metabolic activity, expression and secretion of prostate specific antigen (PSA), cell cycle profile, and the expression of genes involved in development and progression of prostate cancer. Treatment with a subcytotoxic concentration of ENL (60 μM for 72 h) was found to reduce: cell density (57.5%, SD 7.23, p < 0.001), metabolic activity (55%, SD 0.03, p < 0.001), secretion of PSA (48.50% SD 4.74, p = 0.05) and induce apoptosis (8.33‐fold SD 0.04, p = 0.001) compared to untreated cells. Cotreatment with 10 μM etoposide was found to increase apoptosis by 50.17% (SD 0.02, p < 0.001). Additionally, several key genes (e. g. MCMs, survivin and CDKs) were beneficially regulated by ENL treatment (p < 0.05). The data suggest that the antiproliferative activity of ENL is a consequence of altered expression of cell cycle associated genes and provides novel molecular evidence for the antiproliferative properties of a pure lignan in prostate cancer.     

The red clover isoflavone irilone is largely resistant to degradation by the human gut microbiota

Intestinal bacteria may influence bioavailability and physiological activity of dietary isoflavones. We therefore investigated the ability of human intestinal microbiota to convert irilone and genistein in vitro. In contrast to genistein, irilone was largely resistant to transformation by fecal slurries of ten human subjects. The fecal microbiota converted genistein to dihydrogenistein, 6′‐hydroxy‐O‐desmethylangolensin, and 2‐(4‐hydroxyphenyl)‐propionic acid. However, considerable interindividual differences in the rate of genistein degradation and the pattern of metabolites formed from genistein were observed. Only one metabolite, namely dihydroirilone, was formed from irilone in minor amounts. In further experiments, Eubacterium ramulus, a prevalent flavonoid‐degrading species of the human gut, was tested for transformation of irilone. In contrast to genistein, irilone was not converted by E. ramulus. Irilone only differs from genistein by a methylenedioxy group attached to the A‐ring of the isoflavone skeleton. This substitution obviously restricts the degradability of irilone by human intestinal bacteria.     

Induction of cell apoptosis in 3T3-L1 pre-adipocytes by flavonoids is associated with their antioxidant activity

Obesity is biologically characterized at the cellular level by an increase in the number and size of adipocytes differentiated from fibroblastic pre-adipocytes in adipose tissue. In this study, we focused on the relationship between the influence of flavonoids on cell population growth and their antioxidant activity. The results showed that the inhibition of flavonoids (naringenin, rutin, hesperidin, resveratrol, naringin and quercetin) on 3T3-L1 pre-adipocytes was 28.3, 8.1, 11.1, 33.2, 5.6 and 71.5%, respectively. In oxygen radical absorbance capacity (ORAC) assay, quercetin had the highest ORAC(ROO) value among the six flavonoids tested. Apoptosis assays showed that quercetin increased apoptotic cells in time- and dose-dependent manner. Treatment of cells with quercetin decreased the mitochondrial membrane potential in the courses of time and dose. The cell apoptosis/necrosis assay showed that quercetin increased the number of apoptotic cells, but not necrotic cells. Quercetin treatment of cells caused a significant time- and dose-dependent increase in the caspase-3 activity. Western analysis indicated that treatment of quercetin markedly down-regulated PARP and Bcl-2 proteins, and activated caspase-3, Bax, and Bak proteins. These results indicate that quercetin efficiently inhibits cell population growth and induction of apoptosis in 3T3-L1 pre-adipocytes.     

Sulforaphane inhibits smooth muscle cell proliferation and migration by reducing MMP-9 activity via the Ras and RhoA/ROCK pathways

Vascular smooth muscle cell (VSMC) proliferation and migration triggered by inflammatory stimuli are involved in the development and progression of atherosclerosis. Here, we investigate the capacity of sulforaphane (SFN) on TNF-α-induced proliferation, migration and signal transduction in A7r5 smooth muscle cells. SFN inhibited the migration ability of A7r5 cells induced by TNF-α using wound healing assay and Boyden chamber assay. After treatment of SFN at various concentrations with TNF-α, A7r5 cell reduced matrix metalloproteinase-9 (MMP-9) expression. Fluorescent phalloidin staining demonstrated that increased intense F-actin staining in the TNF-α-induced A7r5 cells was inhibited by SFN. The levels of Ras and RhoA/ROCK-related proteins in the TNF-α treated A7r5 cells were increased, whereas these protein expression were attenuated by SFN. The results suggested that SFN could mediate A7r5 cells migration by reducing MMP-9 activity involving the suppression of the Ras and RhoA signaling pathways.     

The modulation of endothelial cell gene expression by green tea polyphenol-EGCG

Human and animal studies have shown that green tea consumption is associated with a reduced risk of some cancers. This has been attributed to its polyphenol components, in particular (-)-epigallocatechin gallate (EGCG). In addition to be a cancer chemopreventive agent, EGCG inhibits angiogenesis, thus reducing tumor growth and metastasis. We tested EGCG modulation on the gene expression profile of endothelial cells stimulated by VEGF using Affymetrix microarrays. A total of 421 genes were up-regulated and 72 genes were down-regulated at the false discovery rate of 5% by VEGF, EGCG, and EGCG pretreatment followed by VEGF stimulation. The changes in the expression of several pivotal genes were validated by real-time PCR. Furthermore, we have identified two signaling pathways (Wnt and Id) involved in cell proliferation were inhibited by EGCG treatment, suggesting the negative regulation of EGCG on cell proliferation. Our results also indicate that the antiangiogenesis effect of EGCG is partially mediated through its broad inhibition on endothelial cell proliferation. Our data further support earlier observations that the anticancer effect of EGCG is mediated through changes in the expression of genes that are associated with cell proliferation.