Chondroitin sulfate is not digested at all in the mouse small intestine but may suppress interleukin 6 expression induced by tumor necrosis factor-α.

Salmon nasal cartilage proteoglycan (PG) was orally administered to mice. The PG digest was recovered from the small intestine, and its sugar chain size and unsaturated disaccharide content were examined. The elution position of the PG digest following Sepharose CL-4B chromatography was consistent with that of actinase-digested PG prior to administration. The PG digest was incubated with chondroitinase ABC, which resulted in the elution pattern of the unsaturated disaccharides being identical to that of the degraded product of actinase-digested PG. The core protein of PG was digested in the mouse small intestine, but chondroitin sulfate, which is the sugar chain of PG, was not degraded at all. Then, the effects of chondroitin 4- and 6-sulfates on human colon cancer cells were examined. These chondroitin sulfates were found to suppress the expression of interleukin-6 induced by TNF-α. Overall, the chondroitin sulfate chain may act on the intestinal epithelium and suppress inflammation of the intestinal tract.

Antimicrobial properties of L-amino acid oxidase: biochemical features and biomedical applications.

Mucus layer that covers the body surface of various animal functions as a defense barrier against microbes, environmental xenobiotics, and predators. Previous studies have reported that L-amino acid oxidase (LAAO), present in several animal fluids, has potent properties against pathogenic bacteria, viruses, and parasites. LAAO catalyzes the oxidative deamination of specific L-amino acids with the generation of hydrogen peroxide and L-amino acid metabolites. Further, the generated hydrogen peroxide is involved in oxidation (direct effect) while the metabolites activate immune responses (indirect effect). Therefore, LAAO exhibits two different mechanisms of bioactivation. Previously, we described the selective, specific, and local oxidative and potent antibacterial actions of various LAAOs as potential therapeutic strategies. In this review, we focus on their biochemical features, enzymatic regulations, and biomedical applications with a view of describing their probable role as biochemical agents and biomarkers for microbial infections, cancer, and autoimmune-mediated diseases. We consider that LAAOs hold implications in biomedicine owing to their antimicrobial activity wherein they can be used in treatment of infectious diseases and as diagnostic biomarkers in the above-mentioned diseased conditions. KEY POINTS: •Focus on biochemical features, enzymatic regulation, and biomedical applications of LAAOs. •Mechanisms of antimicrobial activity, inflammatory regulation, and immune responses of LAAOs. •Potential biomedical application as an antimicrobial and anti-infection agent, and disease biomarker.

Phosphorylation of Thr328 in hyaluronan synthase 2 is essential for hyaluronan synthesis.

Hyaluronan synthase 2 (HAS2) is an integral membrane protein composed of multi-membrane-spanning regions and a large intracellular loop (HAS2-loop). We previously examined the effect of phorbol 12-myristate 13-acetate (PMA) and/or 4-methylumbelliferone (4-MU) on the synthesis of hyaluronan (HA) in human skin fibroblasts and found that TPA and 4-MU have opposing effects on HA synthesis by phosphorylation and O-linked ß-N-acetylglucosaminylation of HAS2, respectively. In this study, we constructed an expression vector for the HAS2-loop and analyzed its post-translational modification by PMA and 4-MU using mass spectrometry. We identified a phosphorylation site at the position corresponding to the Thr328 position of full-length HAS2, which was detected in the cells regardless of the presence of PMA or 4-MU. We next prepared T328A site-directed mutagenesis construct-transfected cells and investigated HA synthesis. The amount of HA was increased in cells expressing full-length HAS2 compared to in mock cells, whereas the amount of HA synthesized by cells transfected with the T328A site-directed mutagenesis construct was the same as that in mock cells. This phosphorylation site corresponded with the casein kinase 1 substrate motif. These results suggest that Thr328 phosphorylation is an essential factor for HA synthesis by HAS2 and the role of HAS2-loop may be useful in analyzing the regulation of HAS2 synthesis in physiological and pathological conditions.

Metal coordination by L-amino acid oxidase derived from flounder Platichthys stellatus is structurally essential and regulates antibacterial activity.

L-amino acid oxidases (LAAOs) have antibacterial activity and play important roles in innate immunity. We have previously identified a LAAO of ~52 kDa in size from the mucus layer of the flounder Platichthys stellate (psLAAO1) and have successfully produced psLAAO1 as a secreted bioactive recombinant protein by using Pichia pastoris (P. pastoris). The recombinant psLAAO1 inhibited the growth of bacteria to the same levels as native psLAAO1 present in the mucus layer. In this study, homology modeling of psLAAO1 predicted metal coordination by residues Y241, H348, and D406. We show that the Michaelis constant (Km) of psLAAO1 decreased and the catalytic constant (Kcat/Km) value increased following pre-treatment of the protein with a chelating agent. In contrast to the non-chelated protein sample, enzymatic activity of EDTA-treated psLAAO1 gradually decreased or was absent after one or two freeze-thaw cycles. The H348A psLAAO1 mutant generated by site-directed mutagenesis and recombinantly produced by P. pastoris did not display antibacterial activity. The results of the metal detection assay revealed that for the non-metal coordinating histidine mutant (H209A, control), the levels of iron, zinc, and magnesium were similar to those of wild-type psLAAO1, whereas magnesium was not detected in the H348A mutant sample. A wild-type psLAAO1 sample treated with chelating agent did not contain zinc and magnesium ions. In conclusion, metal coordination by psLAAO1 affects enzymatic activity, and H348 is involved in the coordination of magnesium, and metal coordination by psLAAO1 provides essential structural stability. KEY POINTS: • Homology modeling of psLAAO1 predicted metal coordination by residue H348 • The H348A psLAAO1 mutant showed no antibacterial activity or magnesium coordination • Metal coordination by H348 affects enzyme activity and structural stability.

Structural change of retinoic-acid receptor-related orphan receptor induced by binding of inverse-agonist: Molecular dynamics and ab initio molecular orbital simulations.

To elucidate structural changes in the retinoic acid receptor-related orphan receptor gamma (RORγt) induced by the binding of an agonist or an inverse agonist, we conducted molecular dynamics (MD) simulations in explicit water. In addition, ab initio fragment molecular orbital calculations were carried out for certain characteristic structures obtained from the MD simulations to reveal important interactions between the amino acid residues of RORγt, and to distinguish the different effects in the binding of an agonist and an inverse agonist on the structure of RORγt. The results elucidate that the hydrogen bond between His479 of helix11 (H11) and Tyr502 of helix12 (H12) is important to keep the H12 conformation in the agonist-bound RORγt. In contrast, in the inverse-agonist-bound RORγt, the side chain of His479 rotates, significantly weakening the interaction between His479 and Tyr502, leading to a conformational change in H12. Therefore, the present molecular simulations clearly indicate that the conformational change in the side chain of His479 in the inverse-agonist-bound RORγt is the main reason for the H12 destabilization induced by the binding of the inverse agonist. Such a conformational change does not occur on the binding of the agonist in RORγt, owing to the strong hydrogen bond between His479 and Tyr502.

Dermatan sulfate oligosaccharides having reducing end 2, 5-anhydro-d-talose inhibit bovine testicular hyaluronidase activity.

Dermatan sulfate oligosaccharides having reducing end 2,5-anhydro-d-talose were prepared by partial N-deacetylation of dermatan sulfate polysaccharide with hydrazine followed by deamination with nitrous acid, and the effect of these oligosaccharides on the activity of bovine testicular hyaluronidase was investigated. Hydrolysis activity and transglycosylation activity of this enzyme were assessed in an independent reaction system by analyzing the products by HPLC. Dermatan sulfate oligosaccharides inhibited hyaluronan hydrolysis by bovine testicular hyaluronidase. Kinetic analysis of the hydrolysis reaction revealed that the inhibition mode by dermatan sulfate oligosaccharides was as competitive as that previously shown by chondroitin sulfate oligosaccharides. Transglycosylation of hyaluronan by bovine testicular hyaluronidase, as a reverse reaction of hydrolysis of hyaluronan, was also inhibited. These inhibitory effects were dependent on the dose and sulfation degree of dermatan sulfate.

Change in binding states between catabolite activating protein and DNA induced by ligand-binding: molecular dynamics and ab initio fragment molecular orbital calculations.

The transcription mechanism of genetic information from DNA to RNA is efficiently controlled by regulatory proteins, such as catabolite activator protein (CAP), and their ligands. When cyclic AMP (cAMP) binds to CAP, the complex forms a dimer and binds specifically to DNA to activate the transcription mechanism. On the other hand, when cyclic GMP (cGMP) binds to CAP, the complex has no marked effect on the mechanism. In our previous study, based on molecular dynamics (MD) and ab initio fragment molecular orbital (FMO) methods, we elucidated which residues of CAP are important for the specific interactions between CAP and DNA in the CAP-monomer+DNA + cAMP complex. However, this monomer model for CAP cannot describe real interactions between the CAP-dimer and DNA because CAPs form a dimer before binding to DNA. Accordingly, here, we investigated stable structures and their electronic states for the CAP-dimer+DNA complex with cAMP or cGMP ligand, to clarify the influence of ligand-binding on the interactions between CAP-dimer and DNA. The MD simulations elucidated that the DNA-binding domains of CAP-dimer behave differently depending on the ligand bound to the CAP-dimer. In addition, FMO calculations revealed that the binding energy between CAP-dimer and DNA for the CAP-dimer+DNA + cAMP complex is larger than that for the CAP-dimer+DNA + cGMP complex, being consistent with experiments. It was also highlighted that the Arg185 and Lys188 residues of CAP-dimer are important for the binding between CAP-dimer and DNA. These results provide useful information for proposing new compounds that efficiently control the transcription mechanism.

Increase in muscle endurance in mice by dietary Yamabushitake mushroom (Hericium erinaceus) possibly via activation of PPARδ.

Skeletal muscle fiber is largely classified into two types: type 1 (slow-twitch) and type 2 (fast-twitch) fibers. Meat quality and composition of fiber types are thought to be closely related. Previous research showed that overexpression of constitutively active peroxisome proliferator-activated receptor (PPAR)δ, a nuclear receptor present in skeletal muscle, increased type 1 fibers in mice. In this study, we found that hexane extracts of Yamabushitake mushroom (Hericium erinaceus) showed PPARδ agonistic activity in vitro. Eight-week-old C57BL/6J mice were fed a diet supplemented with 5% (w/w) freeze-dried Yamabushitake mushroom for 24 hr. After the treatment period, the extensor digitorum longus (EDL) muscles were excised. The Yamabushitake-supplemented diet up-regulated the PPARδ target genes Pdk4 and Ucp3 in mouse skeletal muscles in vivo. Furthermore, feeding the Yamabushitake-supplemented diet to mice for 8 weeks resulted in a significant increase in muscle endurance. These results indicate that Yamabushitake mushroom contains PPARδ agonistic ligands and that dietary intake of Yamabushitake mushroom could activate PPARδ in skeletal muscle of mice. Unexpectedly, we observed no significant alterations in composition of muscle fiber types between the mice fed control and Yamabushitake-supplemented diets.

Regulation of radiosensitivity by 4-methylumbelliferone via the suppression of interleukin-1 in fibrosarcoma cells.

Tumor recurrence and distant metastasis following radiotherapy, which can lead to poor prognosis, are caused by residual cancer cells that acquire radioresistance. Chemotherapy or a combination of targeted inhibitors can potentially enhance radiation sensitivity and prevent metastasis. It was previously reported that co-administration of the hyaluronan synthesis inhibitor 4-methylumbelliferone (4-MU) enhanced the lethality of X-ray irradiation in HT1080 human fibrosarcoma cells and decreased their invasiveness to a greater extent than either treatment alone. To clarify the molecular basis of these effects, the present study conducted mRNA expression profiling by cDNA microarray to identify the signaling pathways that are altered under this combination treatment. The activation state of the signaling pathways was classified by z-scores in the Ingenuity Pathway Analysis. The results revealed that the pro-inflammatory cytokines interleukin (IL)-6 and IL-8 were activated by 2 Gy X-ray irradiation, an effect that was abolished by co-administration of 4-MU. Similar trends were observed for the upstream signaling component IL-1. These results indicate that the radiosensitivity of fibrosarcoma cells is improved by suppressing inflammation through the administration of 4-MU.

Proteoglycan-substrate gel zymography for the detection of chondroitin sulfate-degrading enzymes.

Chondroitin sulfate (CS), a linear polysaccharide, is a major component of the cartilage matrix. Although CS plays various roles in several biological and pathological processes, most details regarding its metabolism are still poorly understood. Some CS-degrading enzymes have been identified in mammals, but their expression patterns and localizations remain unclear. Here we present a simple zymography procedure to detect CS-degrading enzymes using salmon nasal cartilage proteoglycans as substrates. This method should be useful to explore CS-degrading enzymes.

Blackcurrant Anthocyanins Increase the Levels of Collagen, Elastin, and Hyaluronic Acid in Human Skin Fibroblasts and Ovariectomized Rats.

Blackcurrants (Ribes nigrum L.) contain high levels of anthocyanin polyphenols, which have beneficial effects on health, owing to their antioxidant and anticarcinogenic properties. Phytoestrogens are plant-derived substances with estrogenic activity, which could have beneficial effects on the skin. Estradiol secretion decreases during menopause, reducing extracellular matrix (ECM) component production by skin fibroblasts. Using a normal human female skin fibroblast cell line (TIG113) and ovariectomized rats, the present study investigated whether an anthocyanin-rich blackcurrant extract (BCE) and four blackcurrant anthocyanins have novel phytoestrogenic activities that could benefit the skin in menopausal women. In TIG113 cells, a microarray and the Ingenuity® Pathway Analysis showed that 1.0 µg/mL of BCE upregulated the expression of many estrogen signaling-related genes. A quantitative RT-PCR analysis confirmed that BCE (1.0 or 10.0 µg/mL) and four types of anthocyanins (10 µM) altered the mRNA expression of ECM proteins and enzymes involved in ECM turnover. Immunofluorescence staining indicated that the anthocyanins stimulated the expression of ECM proteins, such as collagen (types I and III) and elastin. Dietary administration of 3% BCE to ovariectomized rats for 3 months increased skin levels of collagen, elastin, and hyaluronic acid. This is the first study to show that blackcurrant phytoestrogens have beneficial effects on skin experimental models.

Serum miR-375-3p increase in mice exposed to a high dose of ionizing radiation.

Exposure to high-doses of ionizing radiation (IR) leads to development of a strong acute radiation syndrome (ARS) in mammals. ARS manifests after a latency period and it is important to develop fast prognostic biomarkers for its early detection and assessment. Analysis of chromosomal aberrations in peripheral blood lymphocytes is the gold standard of biological dosimetry, but it fails after high doses of IR. Therefore, it is important to establish novel biomarkers of exposure that are fast and reliable also in the high dose range. Here, we investigated the applicability of miRNA levels in mouse serum. We found significantly increased levels of miR-375-3p following whole body exposure to 7 Gy of X-rays. In addition, we analyzed their levels in various organs of control mice and found them to be especially abundant in the pancreas and the intestine. Following a dose of 7 Gy, extensive cell death occurred in these tissues and this correlated negatively with the levels of miR-375-3p in the organs. We conclude that high expressing tissues of miR-375-3p may secrete this miRNA in serum following exposure to 7 Gy. Therefore, elevated miR-375-3p in serum may be a predictor of tissue damage induced by exposure to a high radiation dose.

Chondroitin sulfate proteoglycans from salmon nasal cartilage inhibit angiogenesis.

Because cartilage lacks nerves, blood vessels, and lymphatic vessels, it is thought to contain factors that inhibit the growth and development of those tissues. Chondroitin sulfate proteoglycans (CSPGs) are a major extracellular component in cartilage. CSPGs contribute to joint flexibility and regulate extracellular signaling via their attached glycosaminoglycan, chondroitin sulfate (CS). CS and CSPG inhibit axonal regeneration; however, their role in blood vessel formation is largely unknown. To clarify the function of CSPG in blood vessel formation, we tested salmon nasal cartilage proteoglycan (PG), a member of the aggrecan family of CSPG, for endothelial capillary-like tube formation. Treatment with salmon PG inhibited endothelial cell adhesion and in vitro tube formation. The anti-angiogenic activity was derived from CS in the salmon PG but not the core protein. Salmon PG also reduced matrix metalloproteinase expression and inhibited angiogenesis in the chick chorioallantoic membrane. All of these data support an anti-angiogenic role for CSPG in cartilage.

Anthocyanin­rich blackcurrant extract inhibits proliferation of the MCF10A healthy human breast epithelial cell line through induction of G0/G1 arrest and apoptosis.

Blackcurrants (Ribes nigrum L., Grossulariaceae) possess a high content of anthocyanin polyphenols, which have been demonstrated to exhibit beneficial effects on health due to their antioxidant and anticarcinogenic prope-rties. The present study investigated novel functions of anthocyanin­rich blackcurrant extracts (BCEs) in a healthy mammary epithelial cell line, MCF10A. The percentages of viable cells were 85, 75, 53 and 31% following exposure to 50, 100, 200 and 400 µg/ml BCE, respectively. The half­maximal response concentration of BCE was 237.7 µg/ml. Microarray and Ingenuity® Pathway Analysis demonstrated that BCE downregulated cell cycle signaling, including upstream genes with mitotic roles such as polo­like kinase signaling. BCE increased the number of cells in the G0/G1 phase and decreased the number of cells in the S and G2/M phases. Alkaline comet assays demonstrated that 50 and 100 µg/ml BCE induced DNA damage in a dose­dependent manner. Cultures treated with 0, 50, and 100 µg/ml BCE contained 4.6, 13.4 and 16.0% apoptotic cells, respectively. As compared with the untreated cultures (1.9%), the number of necrotic cells increased in the 100 µg/ml BCE­treated cultures (from 1.9 to 4.3%) but not in the 50 µg/ml BCE­treated cultures. Reverse transcription­quantitative polymerase chain reaction analysis demonstrated that BCE reduced mRNA expression of the genomic caretaker lysine­specific demethylas  5B (KDM5B). The results suggested that blackcurrant anthocyanins may act as cell arrest and death inducers via KDM5B downregulation in healthy breast cells.

Impact of time interval and dose rate on cell survival following low-dose fractionated exposures.

Enhanced cell lethality, also known as hyper-radiosensitivity, has been reported at low doses of radiation (≤0.5 Gy) in various cell lines, and is expected to be an effective cancer therapy. We conducted this study to examine the impact of time interval and dose rate of low-dose fractionated exposures with a short time interval. We evaluated the cell-survival rates of V79 and A549 cells using clonogenic assays. We performed fractionated exposures in unit doses of 0.25, 0.5, 1.0 and 2.0 Gy. We exposed the cells to 2 Gy of X-rays (i) at dose-rates of 1.0, 1.5 and 2.0 Gy/min at 1-min intervals and (ii) at a dose-rate of 2.0 Gy/min at 10-s, 1-min and 3-min intervals by fractionated exposures. Apoptosis and cell cycle analyses were also evaluated in the fractionated exposures (unit dose 0.25 Gy) and compared with single exposures by using flow cytometry. Both cell-type survival rates with fractionated exposures (unit dose 0.25 Gy) with short time intervals were markedly lower than those for single exposures delivering the same dose. When the dose rates were lower, the cytotoxic effect decreased compared with exposure to a dose-rate of 2.0 Gy/min. On the other hand, levels of apoptosis and cell cycle distribution were not significantly different between low-dose fractionated exposures and single exposures in either cell line. These results indicate that a stronger cytotoxic effect was induced with low-dose fractionated exposures with a short time interval for a given dose due to the hyper-radiosensitivity phenomenon, suggesting that dose rates are important for effective low-dose fractionated exposures.

UDP/P2Y6 receptor signaling regulates IgE-dependent degranulation in human basophils.

BACKGROUND: P2Y purinergic receptors (P2YR) are G protein-coupled receptors that are stimulated by extracellular nucleotides. They mediate cellular effects by regulating cAMP production, protein kinase C activation, inositol trisphosphate generation, and Ca2+ release from intracellular stores. The P2Y6 receptor of this family is selectively stimulated by UDP, and selectively inhibited by MRS2578. In the present study, we examined the effect of UDP/P2Y6 receptor signaling on IgE-dependent degranulation in human basophils. METHODS: Basophils were purified from human peripheral blood. The mRNA expression of genes encoding P2YR and ecto-nucleoside triphosphate diphosphohydrolase (ENTPDase) was measured by RT-PCR. Intracellular Ca2+ influx via UDP/P2Y6 receptor signaling in basophils was detected using a calcium probe. The effect of UDP/P2Y6 receptor signaling on IgE-dependent degranulation in basophils was confirmed by measuring CD63 expression by flow cytometry. Autocrine secretion of nucleotides was detected by HPLC analysis. RESULTS: We showed that purified basophils express P2Y6 mRNA and that UDP increased intracellular Ca2+, which was reduced by MRS2578 treatment. UDP promoted IgE-dependent degranulation. Furthermore, MRS2578 inhibited IgE-dependent degranulation in basophils. HPLC analysis indicated that basophils spontaneously secrete UTP. In addition, basophils expressed the extracellular nucleotide hydrolases ENTPDase2, ENTPDase3, and ENTPDase8. CONCLUSIONS: This study showed that UDP/P2Y6 receptor signaling is involved in the regulation of IgE-dependent degranulation in basophils, which might stimulate the P2Y6 receptor via the autocrine secretion of UTP. Thus, this receptor represents a potential target to regulate IgE-dependent degranulation in basophils during allergic diseases.

Anti-tumor and anti-invasion effects of a combination of 4-methylumbelliferone and ionizing radiation in human fibrosarcoma cells.

Hyaluronan (HA) is a major component of the extracellular matrix that is synthesized in excess in cancer tissues. 4-methylumbelliferone (MU) inhibits the synthesis of HA and is closely related to the invasion and metastasis of cancer. However, the effects of MU in conjunction with cancer radiotherapy remain unknown. The present study assessed the anti-tumor and anti-invasion effects of the concomitant use of ionizing radiation (IR) and 100 µM MU on human fibrosarcoma HT1080 cells. Cell viability and cellular invasion potency assays were performed. There was a greater decrease in the viability of cells cultured with a combination of 2 Gy IR and MU compared with untreated control cells. In addition, cell cycle distribution analysis demonstrated that a higher proportion of these cells were in the sub-G1 phase and higher fractions of annexin-V positive, propidium iodide positive cells (i.e., apoptotic cells) were observed. HA concentration in the 2 Gy irradiated culture was similar to that in the non-irradiated control culture, however, it significantly decreased following the administration of both MU alone and 2 Gy IR with MU. Furthermore, treatment with 2 Gy IR and MU resulted in a significant decrease in the invasion rate and matrix metalloproteinase (MMP)-2 and MPP-9 expression. Taken together, these results suggest that the administration of MU with 2 Gy IR is effective at reducing HA production, cell invasion and the metastatic potential of cancer cells.

Exosomes derived from SW480 colorectal cancer cells promote cell migration in HepG2 hepatocellular cancer cells via the mitogen-activated protein kinase pathway.

Exosomes are membrane-derived extracellular vesicles that have recently been recognized as important mediators of intercellular communication. In the present study, we investigated the effects of exosomes derived from SW480 colorectal cancer cells in recipient HepG2 hepatocellular cancer cells. We demonstrated that SW480-derived exosomes were taken up by the recipient HepG2 cells via dynamin-dependent endocytosis and were localized to the HepG2 lysosomes. In addition, SW480-derived exosomes induced the phosphorylation of extracellular signal-regulated kinase (ERK)1/2 following their uptake into HepG2 cells. Of note, these changes occurred during the early phase after exosome treatment. Furthermore, SW480-derived exosomes promoted the migration of recipient HepG2 cells in a wound-healing assay, which was suppressed by pretreatment with U0126, an upstream inhibitor of ERK1/2. These results indicated that SW480-derived exosomes activated a classical mitogen-activated protein kinase pathway in recipient HepG2 cells via dynamin-dependent endocytosis and subsequently enhanced cell migration by ERK1/2 activation. Our results provide new insights into the regulation of cellular functions by exosomes.

Phytoestrogenic activity of blackcurrant (Ribes nigrum) anthocyanins is mediated through estrogen receptor alpha.

SCOPE: Blackcurrants (Ribes nigrum L., Grossulariaceae) contain high amounts of anthocyanin polyphenols, which have antioxidant and anti-carcinogenic health benefits. This study analyzed the potential phytoestrogenic effects of blackcurrant extract (BCE) in breast cancer (MCF-7) and human endometrial cancer (Ishikawa) cell lines that over-express estrogen receptor alpha (ERα), as well as in immature female rats. METHODS AND RESULTS: Microarray analysis and Ingenuity® Pathway Analysis showed that BCE activated the ERα pathway, whereas quantitative-PCR confirmed that BCE and four types of anthocyanins up-regulated genes downstream of ERα. BCE (0.1-1.0 µg/mL) and anthocyanins (0.1-10 µM) induced MCF-7 cell proliferation; however, this effect was blocked by ER antagonist fulvestrant. Flow cytometry showed that anthocyanins reduced and increased the number of MCF-7 cells in the G0/G1 and G2/M phases, respectively. Anthocyanins stimulated ERα transcriptional activity in human ERα reporter assays and induced alkaline phosphatase activity in Ishikawa cells. Competition assays and in silico analysis indicated that anthocyanins bind to ERα. Finally, BCE focally induced stratification of columnar epithelial cells in the rat uterus and increased cytoplasmic mucin levels in these cells. CONCLUSION: These results suggest that blackcurrant anthocyanins act as phytoestrogens in vitro and in vivo.