Search

Background/Aims: Calycosin, a phytoestrogenic compound, has recently emerged as a promising antitumor drug. It has been shown that calycosin suppresses growth and induces apoptosis of breast cancer cells. However, the effect of calycosin on migration and invasion of breast cancer cells and the underlying molecular mechanisms have not been elucidated. Methods: Human breast cancer cells MCF-7 and T47D were treated with, or without, different doses (0, 6.25, 12.5, 25, 50, 100 or 150 μM) of calycosin, and the viability of different groups was determined by MTT assay. Next, the inhibitory effect of higher doses (50, 100 or 150 μM) of calycosin on migration and invasion of the two cell lines was determined by wound healing and transwell assay. The relative expression levels of forkhead box P3 (Foxp3), vascular endothelial growth factor (VEGF) and matrix metalloproteinase-9 (MMP-9) in MCF-7 and T47D cells were determined by quantitative RT-PCR and Western blot. Results: Treatment with lower doses (6.25 or 12.5 μM) promoted proliferation of breast cancer cells, but with higher doses significantly reduced the viability of MCF-7 and T47D cells. Furthermore, higher doses of calycosin were found to inhibit migration and invasion of the two cell lines in a dose-dependent manner. Additionally, treatment with a higher dose of calycosin significantly reduced the expression levels of Foxp3, followed by down-regulation of VEGF and MMP-9 in both MCF-7 and T47D breast cancer cells. Conclusion: Treatment with a higher dose of calycosin tends to reduce migration and invasion capacity of human breast cancer cells, by targeting Foxp3-mediated VEGF and MMP-9 expression.

Background/Aims: Calycosin is a bioactive component of Astragali Radix, a Chinese herb for treating allergy. We have previously demonstrated that calycosin effectively inhibited allergic inflammation efficiently. The aim of this study was to explore the mechanism of calycosin on epithelial cells in allergic inflammation. Methods: An initial stage of atopic dermatitis (AD) model in which mice were just sensitized with FITC, was established in vivo and immortalized human keratinocytes (HaCaT cells) were utilized in vitro. Initiative key cytokines, TSLP and IL-33, were measured by ELISA, qPCR, immunofluorescence and Western blot. The junctions in epithelial cells were observed by electron microscopy and tight junctions (TJs) (Occludin and ZO-1) were assessed by Western blot and immunofluorescence. TLR4, MyD88, TAK1, TIRAP and NF-κB were measured by qPCR or Western blot. Results: The results showed that TSLP and IL-33 were inhibited significantly by calycosin in the initial stage of AD model. Simultaneously, calycosin attenuated the separated gap among the epithelial cells and increased the expression of TJs. TSLP/IL-33 and TJs were similarly affected in LPS-stimulated HaCaT cells in vitro. Meanwhile, calycosin not only inhibited the expressions of TLR4, MyD88, TAK1 and TIRAP, but also reduced NF-κB activation in vitro and in vivo. An NF-κB inhibitor enhanced the expressions of TJs and reduced that of TSLP/IL-33 in LPS-stimulated HaCaT cells. Conclusion: These results indicated that calycosin reduced the secretion of TSLP/IL-33 and attenuated the disruption of epithelial TJs by inhibiting TLR4 mediated NF-κB signaling pathway. These findings help to understand the beneficial effects of calycosin on AD, and to develop effective preventive or therapeutic strategies to combat this disease and other epithelial barrier deletion-mediated allergic diseases.

Background: 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. Methods: 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. Results: 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. Conclusion: Calycosin is more effective in inhibiting breast cancer growth in comparison with genistein, through its regulation of Akt signaling pathways and HOTAIR expression.

Background: Previous studies have shown that some phytoestrogens inhibits proliferation and induces apoptosis in estrogen-dependent cancers via estrogen receptor (ER)-mediated signaling pathway. In view of the expression of ER in human osteosarcoma cells, the purpose of this study is to investigate whether formononetin and calycosin, two of the major isoflavones in Radix astragali, could also elicit anti-tumor activity against osteosarcoma, along with the underlying mechanism. Methods: Human osteosarcoma cells U2OS were respectively treated with various concentrations of formononetin or calycosin. Cell proliferation was determined by MTT assay, while apoptosis by flow cytometry. Next, the expression levels of apoptosis-related genes ERK, Akt, Bcl-2, Bax and caspase-3 were quantified by real-time PCR and Western blotting. Results: Formononetin exhibited higher anti-proliferative activities toward human osteosarcoma cells U2OS, when compared with calycosin. Therefore, U2OS cells were then respectively treated with various concentrations of formononetin, in order to elucidate the isoflavones-related signaling pathway. It was found that formononetin dose-dependently triggered apoptosis of U2OS cells in vitro. Furthermore, treatment of formononetin led to significant inactivation of ERK and Akt, followed by downregulation of Bcl-2, upregulation of Bax and finally increased expression of caspase-3. Conclusion: Formononetin is more effective than calycosin at promoting cell death of U2OS cells by induction of apoptosis, which is mediated by inactivation of ERK and Akt signaling pathways. Thus isoflavones, especially formononetin, may be useful as anti-cancer drugs for osteosarcoma through their apoptosis-inducing effects.

Background: Calycosin and formononetin are two main components of isoflavones. In our previous studies, we have respectively reported their antitumor activities on breast cancer cell MCF-7. To further investigate the feasibility of isoflavones in clinically treating breast carcinoma, here we specifically focused on the comparison between calycosin and formononetin, along with the relevant mechanism. Methods: ER-positive (MCF-7, T-47D) and ER-negative breast cancer cells (MDA-231, MDA-435) were respectively treated with calycosin or formononetin. Cell proliferation and apoptosis were measured by MTT assay and flow cytometry. mRNA levels of ER beta (ERβ) and miR-375 were quantifed by real-time PCR. Expression of ERβ and insulin-like growth factor 1 receptor (IGF-1R), and activation of poly (ADP-ribose) polymerase 1 (PARP-1) were determined by Western blotting. Results: Both calycosin and formononetin impaired proliferation and triggered apoptosis of ER-positive breast cancer cells (MCF-7, T-47D) in a time- and dose-dependent manner, especially in the treatment with calycosin. However, no such effect was observed in ER-negative breast cancer cells, indicating the correlation between isoflavones-induced inhibitory effect and ERs. Thus calycosin and most sensitive MCF-7 cells were used to study the relevant signaling pathway. After the treatment of calycosin, ERβ expression was significantly increased in MCF-7 cells, followed by decrease of IGF-1R, activation of PARP-1 cleavage and downregulation of miR-375. Conclusion: Calycosin has an advantage on inhibiting breast cancer growth in comparison with formononetin, which is obtained by ERβ-mediated regulation of IGF-1R signaling pathways and miR-375 expression.