phytoestrogens and 7-3--dihydroxy-4--methoxyisoflavone

Danggui Buxue Tang (DBT) is a classical Chinese herbal decoction containing two herbs, Astragali Radix (AR) and Angelicae Sinensis Radix (ASR), which serves as dietary supplement for treating women menopausal syndromes. Pharmacological studies indicate that DBT has estrogenic, erythropoietic and osteogenic properties; however, the action mechanism for this complex herbal decoction is not known. Calycosin, a major flavonoid in AR, shares similar structure with β-estradiol, and thus which is hypothesized to be the critical compound of DBT. Here, we aim to investigate the role of calycosin in DBT in terms of its biological functions by using a calycosin-depleted DBT decoction (DBT(Δcal)). The biological functions of DBT(Δcal) and parental DBT were systematically compared.. In order to standardize DBT decoction, four chemical markers were determined and quantified by HPLC. A semi-preparative HPLC method was utilized to prepare DBT(Δcal). The authenticity of DBT(Δcal) was evaluated by LC-QQQ-MS/MS. To reveal the effect of calycosin on DBT functions, several cell assays related to the known properties of DBT were revealed, including estrogenic, erythropoietic and osteogenic functions.. As compared to parental DBT, the estrogenic, erythropoietic and osteogenic abilities were markedly reduced in DBT(Δcal). However, calycosin alone did not show significant responses.. Our results suggest that calycosin is a bioactive chemical in DBT decoction, and which could play a key linker in orchestrating multi-components of DBT as to achieve maximal functions. These discoveries should be invaluable in drug development and in investigating the modernization of traditional Chinese medicine from a new perspective.

Topics: Alkaline Phosphatase; Angelica sinensis; Astragalus propinquus; Cell Differentiation; Cell Line, Tumor; Drugs, Chinese Herbal; HEK293 Cells; Humans; Isoflavones; Phytoestrogens; Plant Roots

Calycosin, which is a kind of typical phytoestrogen, can bind with estrogen receptor and produce estrogen-like effects. Calycosin were reported to have antioxidant, anti-osteoporosis, anti-tumor and immunomodulating activities. This review covers biological activities and its mechanism of calycosin. It will provide a useful reference for clinical research and rational utilization of monomericompound.

Topics: Animals; Apoptosis; Astragalus Plant; Cell Proliferation; Drugs, Chinese Herbal; Humans; Isoflavones; Phytoestrogens

High intake of phytoestrogen has been reported to be associated with the prevention of colorectal cancer (CRC). Calycosin belongs to the phytoestrogen that has been shown to suppress CRC cells in our previous study. However, its anticancer activity and molecular mechanisms have not been elucidated. In this study, we analyzed the effect of calycosin on the viability and apoptosis of human CRC HCT116 and SW480 cells via MTT assay, flow cytometry assay, and caspase-3/7 activity assay. The protein expressions of estrogen receptor β (ERβ), PTEN, and PI3K/Akt signal pathways were determined by Western blot analysis. And then, the alterations of biological behavior in CRC cells transfected with ERβ siRNA were analyzed. Mouse xenograft models were further performed to detect the antitumor effect in vivo. The results show that calycosin reduces CRC cell viability, induces cell apoptosis, and suppresses xenograft tumor growth. The protein expressions of ERβ and PTEN are significantly upregulated following calycosin treatment, whereas p-AKT/AKT ratio and Bcl-2 level are downregulated. Suppressing ERβ with siRNA partially attenuates the reduction in viability and apoptosis induced by calycosin. Our results indicate that calycosin shows inhibitory effects on CRC cells, which might be obtained by targeting ERβ, upregulating PTEN, and inhibiting the PI3K/Akt signal pathway.

Topics: Animals; Apoptosis; Cell Line, Tumor; Cell Proliferation; Colorectal Neoplasms; Estrogen Receptor beta; Gene Expression Regulation, Neoplastic; Humans; Isoflavones; Mice; Phosphatidylinositol 3-Kinases; Phytoestrogens; Proto-Oncogene Proteins c-akt; PTEN Phosphohydrolase; RNA, Small Interfering; Signal Transduction

Estrogen receptor β (ERβ) is the major ER subtype in hepatic stellate cells (HSCs). Previously we reported phytoestrogen calycosin suppressed liver fibrosis progression and inhibited HSC-T6 cell functions, suggesting the effects may be related to ERβ. Here, we explore the effect of overexpressed ERβ on human HSCs and the role of ERβ in pharmacological action of calycosin.. LX-2 cells were transfected with lentivirus to overexpress ERβ. In the presence or absence of overexpressed ERβ, the effects of ERβ and calycosin on proliferation, migration, activation, collagen production and degradation of TGF-β1-induced LX-2 cells and the role of ERβ in the inhibition effect of calycosin were investigated. LX-2 cells overexpressed with ERβ or treated with ER non-selective antagonist ICI182,780 were used to investigate the regulation of ERβ on JAK2/STAT3 signaling pathway. CCK-8 method was used to screen effective doses of calycosin and investigate cell proliferation. The cell migration was detected by transwell chamber assay. The expression of α-SMA was detected by immunofluorescence and western blot. The protein expressions of Col-I, MMP1, TIMP1, JAK2, p-JAK2, STAT3 and p-STAT3 were detected by western blot.. ERβ overexpressed lentivirus was successfully transfected into LX-2 cells with high efficiency. Overexpressed ERβ or calycosin alone inhibited the TGF-β1-induced LX-2 cell proliferation and migration, downregulated the protein expressions of α-SMA, Col-I, TIMP-1, p-STAT3 and upregulated MMP-1. Both overexpressed ERβ and calycosin had no significant effect on JAK2, p-JAK2 and STAT3 expressions. ERβ overexpression further enhanced the above effects of calycosin. However, after the cells were treated with ICI182,780, downregulation of STAT3 phosphorylation induced by calycosin was reversed.. ERβ mediated the inhibition of major functions of LX-2 cell possibly by inhibiting the phosphorylation of STAT3, and was an important pathway through which calycosin exerted anti-liver fibrosis effect.

Topics: Cell Proliferation; Estrogen Receptor beta; Fibrosis; Hepatic Stellate Cells; Humans; Isoflavones; Liver Cirrhosis; Matrix Metalloproteinase 1; Phosphorylation; Phytoestrogens; STAT3 Transcription Factor; Tissue Inhibitor of Metalloproteinase-1; Transforming Growth Factor beta1

Breast cancer is the most commonly diagnosed cancer worldwide. Previously, we reported that calycosin, a typical isoflavone phytoestrogen, triggers apoptosis and is associated with lncRNA HOTAIR in the estrogen receptor (ER)-positive breast cancer MCF-7-cell line. In the present study, we aim to uncover the mechanism of lncRNA HOTAIR in the inhibitory effect induced by calycosin in both ER-positive and ER-negative breast cancer cell lines. Results show that calycosin significantly inhibits proliferation and triggers apoptosis in both ER-positive (MCF-7 and T47D) and ER-negative (MDA-MB-231 and SK-BR-3) breast cancer cell lines, accompanied by downregulation of lncRNA HOTAIR expression. Accordingly, knockdown of lncRNA HOTAIR promotes the anti-tumor effect of calycosin, while overexpression of lncRNA HOTAIR attenuates this effect. Meanwhile, the expression levels of HuR and IGF2BP1 are also reduced by calycosin. More importantly, calycosin facilitates the downregulation of HuR and IGF2BP1 caused by decreasing lncRNA HOTAIR expression, and the upregulation of HuR and IGF2BP1 caused by overexpression of lncRNA HOTAIR is weakened by calycosin. These results demonstrate that downregulating HuR and IGF2BP1 by suppressing lncRNA HOTAIR results in inhibited growth of breast cancer cells by calycosin. In addition, the binding of HuR and IGF2BP1 to lncRNA HOTAIR is detected by RIP assay, implying an interaction between these two proteins and lncRNA HOTAIR. Together, lncRNA HOTAIR may play a carcinogenic role in breast cancer development and has the potential to be a novel therapeutic target for breast cancer in the future, especially in isoflavone phytoestrogen therapy.

Topics: Breast Neoplasms; Cell Line, Tumor; Cell Proliferation; Female; Gene Expression Regulation, Neoplastic; Humans; Isoflavones; Phytoestrogens; RNA, Long Noncoding

Topics: Actins; Animals; Carbon Tetrachloride; Collagen Type I; Estrogen Receptor beta; Isoflavones; Janus Kinase 2; Liver; Liver Cirrhosis; Male; Matrix Metalloproteinase 13; Mice, Inbred C57BL; Phytoestrogens; Protective Agents; STAT3 Transcription Factor; Tissue Inhibitor of Metalloproteinase-1

The present study was designed to investigate the effects of calycosin on hepatic stellate cell (HSC) function and to explore whether the drug exerts its effect through the estrogen receptor. HSC proliferation and migration were measured by MTT assay and transwell chamber assay, respectively. The mRNA and protein expression of α-SMA, COL-I, and ERβ were detected by real-time PCR and Western blotting. The co-localization and expression of α-SMA and ERβ protein were detected by immunofluorescence. All the studies were investigated in the absence or presence of ICI 182,780. The results showed that calycosin inhibited the proliferation of activated HSCs and remarkably inhibited HSC migration. Calycosin significantly reduced the expression of α-SMA and COL-I in activated HSCs. However, with co-treatment with ICI 182,780, the inhibitory effect of calycosin against the above effects was strongly negated. Importantly, calycosin significantly downregulated the expression of ERβ protein, while co-treatment with ICI 182,780 partially reversed the ERβ downregulation. In addition, α-SMA decreased with the decrease of ERβ expression and the subtype of ERβ on HSC is ERβ

Topics: Actins; Cell Line; Cell Movement; Cell Proliferation; Down-Regulation; Estrogen Receptor beta; Hepatic Stellate Cells; Humans; Isoflavones; Phytoestrogens; RNA, Messenger; Transforming Growth Factor beta1

Calycosin and genistein are the two main components of isoflavones. Previously, we reported that these compounds display antitumor activities in the breast cancer cell lines MCF-7 and T47D. In the present study, we investigated the mechanism of action of calycosin and genistein, and their respective efficacies as potential therapies for the treatment of breast carcinoma in the clinic.. MCF-7 cells were treated with calycosin or genistein. Cell proliferation and apoptosis were measured using CCK8 assay and Hoechst 33258. The expression level of phosphorylated Akt protein was determined by western blotting. Expression level of HOTAIR was quantified by real-time PCR.. Both calycosin and genistein inhibited proliferation and induced apoptosis in MCF-7 breast cancer cells, especially after treatment with calycosin. Treatment of MCF-7 cells with calycosin or genistein resulted in decreased phosphorylation of Akt, and decreased expression of its downstream target, HOTAIR.. Calycosin is more effective in inhibiting breast cancer growth in comparison with genistein, through its regulation of Akt signaling pathways and HOTAIR expression.

Topics: Apoptosis; Breast Neoplasms; Cell Line, Tumor; Cell Proliferation; Down-Regulation; Female; Genistein; Humans; Isoflavones; MCF-7 Cells; Phosphorylation; Phytoestrogens; Proto-Oncogene Proteins c-akt; RNA, Long Noncoding; Signal Transduction

Diabetes mellitus (DM) often accompanies liver dysfunction. Astragali Radix is a traditional Chinese herbal medicine that is widely administrated to ameliorate the symptoms of diabetes as well as liver dysfunction, but its acting mechanism is still not yet fully recognized. Advanced glycation end products (AGEs) play a key role in promoting diabetic organ dysfunction. Both hyperglycemia and AGEs can induce insulin resistance, hepatocyte damage and liver dysfunction. We designed this study to explore the effects of the phytoestrogen Calycosin, a major active component of Astragali Radix, on AGEs-induced glucose uptake dysfunction in the hepatocyte cell line and relevant mechanisms. MTT and BrdU methods were applied to evaluate cell viability. 2-NBDG was used to observe glucose uptake by a live cell imaging system. Immunofluorescence method was carried out to investigate GLUT1, GLUT4, and RAGE protein expressions on cell membrane. cAMP content was determined by an EIA method. We found Calycosin concentration-dependently ameliorated AGEs-induced hepatocyte viability damage. AGEs dramatically reduced basal glucose uptake in hepatocytes, and this reduction could be reversed by Calycosin administration. By immunofluorescence detection, we observed that Calycosin could inhibit AGEs-induced GLUT1 expression down-regulation via estrogen receptor (ER). Furthermore, Calycosin decreased AGEs-promoted RAGE and cAMP elevation in hepatocytes. These findings strongly suggest that Calycosin can ameliorate AGEs-promoted glucose uptake dysfunction in hepatocytes; the protection of cell viability and ER-RAGE and GLUT1 pathways play a significant role in this modulation.

Topics: Animals; Astragalus Plant; Biological Transport; Cell Line; Glucose; Glycation End Products, Advanced; Hepatocytes; Isoflavones; Phytoestrogens; Plant Extracts; Rats

Endothelial cell (EC) apoptosis plays a pivotal role in the progression of diabetic complications. Abundant studies have demonstrated the pivotal role of advanced glycation end products (AGEs) on the development of diabetes. The aim of the present study was to investigate the effect of calycosin, a phytoestrogen, on AGEs-induced human umbilical vein endothelial cell (HUVEC) apoptosis. Fluorescence polarization and fluorescence absorption assays indicated that calycosin interacted with AGEs in a time-dependent manner. Further studies found that calycosin entered the cells as detected by HPLC. The MTT method demonstrated that calycosin ameliorated AGEs-induced HUVEC apoptosis in a dose-dependent manner, and statistical significance was observed at 1 × 10(-8) M of calycosin; this behavior was further demonstrated by acridine orange/ethidium bromide staining in that the presence of calycosin dramatically reduced AGEs-induced red staining in HUVECs. Further studies found that pre-incubation with calycosin at 1 × 10(-8) M dramatically increased anti-apoptotic Bcl-2 while decreased pro-apoptotic Bax and Bad expressions as detected by immunocytochemistry, and the effect of calycosin on rebalancing the ratio of Bcl-2/Bax was more significant than that of its glycoside, calycosin-7-O-β-D-glucopyranoside (CG). Furthermore, calycosin slightly reversed AGEs-induced cell oxidative stress at 1 × 10(-8) M, but its antioxidative stress effect was less significant than that of CG. The present study strongly indicates that calycosin can enter the cell and modulate endothelial cell dysfunction by ameliorating AGEs-induced cell apoptosis.

Topics: Antioxidants; Apoptosis; bcl-2-Associated X Protein; bcl-Associated Death Protein; Biological Transport; Cells, Cultured; Dose-Response Relationship, Drug; Glucosides; Glycation End Products, Advanced; Human Umbilical Vein Endothelial Cells; Humans; Isoflavones; Oxidative Stress; Phytoestrogens; Proto-Oncogene Proteins c-bcl-2; Signal Transduction; Time Factors

Calycosin is one of main components in the herb radix astragali and is considered a typical phytoestrogen. It has either estrogenic or antiestrogenic effects that mainly depend on estrogen levels in vivo. This study investigated the effects and mechanisms of calycosin on estrogen receptor (ER)-positive human breast cancer (MCF-7) cells in vitro.. ER-positive MCF-7 cells were treated with different concentrations of calycosin. Effects of calycosin on the proliferation of ER-positive MCF-7 cells were determined by the MTT assay. Apoptosis in these treated cells was examined by flow cytometry. The mRNA and protein levels of Bcl-2 and Bax in these treated cells were also determined by reverse-transcription polymerase chain reaction and immunohistochemical staining, respectively.. Compared with the vehicle control, calycosin stimulated proliferation of ER-positive MCF-7 cells at low concentrations (2, 4, and 8 µmol/L). Furthermore, at these concentrations, calycosin decreased the percentage of early apoptosis in MCF-7 cells, downregulated mRNA and protein levels of Bax, and upregulated those of Bcl-2 at low concentrations. On the other hand, calycosin at higher concentrations (16 and 32 µmol/L) inhibited cell proliferation.. At relatively low concentrations, calycosin has stimulatory effects on the proliferation of MCF-7 cells, with the estrogenic effect the mechanism.

Topics: Astragalus Plant; bcl-2-Associated X Protein; Breast Neoplasms; Cell Line, Tumor; Down-Regulation; Female; Gene Expression; Genes, bcl-2; Humans; Isoflavones; Phytoestrogens; Plant Extracts; Plant Roots; Proto-Oncogene Proteins c-bcl-2; Receptors, Estrogen