7-3--dihydroxy-4--methoxyisoflavone and Breast-Neoplasms

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

It is widely accepted that estrogen can be replaced by phytoestrogens to treat postmenopausal cardiovascular disease and possibly decrease the risk of breast cancer. However, few studies have investigated the effects of phytoestrogens on vascular endothelial cells (ECs). In the present study, we show that the phytoestrogen calycosin (20 μM) stimulated the proliferation of ECs (HUVECs and HMEC-1) but inhibited the growth of breast cancer cells (BCCs) expressing ERα (MCF-7 and T47D). Here we provide evidence for the presence of a positive feedback loop between ERα and long noncoding RNA RP11-65M17.3 in both normal and cancer cells, and calycosin stimulated this feedback loop in ECs but decreased RP11-65M17.3 expression in BCCs. Subsequently, the calycosin-induced activation of this loop decreased the expression of the target of BRIP1 (BRCA1 interacting protein C-terminal helicase 1), increased the phosphorylation of Akt and ERK1/2, and finally inhibited the cleavage of PARP-1 in ECs. In nude mice bearing MCF-7 xenografts, calycosin did not stimulate tumor growth as strongly as 17β-estradiol. Together, these results suggest that calycosin promotes the proliferation of ECs, and notable inhibits the growth of BCCs. A possible reason for these results is the involvement of a feedback loop between ERα and RP11-65M17.3.

Topics: Animals; Breast Neoplasms; Cell Line, Tumor; Cell Proliferation; Estrogen Receptor alpha; Fanconi Anemia Complementation Group Proteins; Feedback, Physiological; Female; Gene Expression Regulation, Neoplastic; Human Umbilical Vein Endothelial Cells; Humans; Isoflavones; Mice; Rats; RNA Helicases; RNA, Long Noncoding; Xenograft Model Antitumor Assays

Topics: Base Sequence; Basic-Leucine Zipper Transcription Factors; Breast Neoplasms; Cell Movement; Cell Proliferation; Disease Progression; Down-Regulation; Epithelial-Mesenchymal Transition; Female; Gene Expression Regulation, Neoplastic; Humans; Isoflavones; MCF-7 Cells; Neoplasm Invasiveness; Neoplasm Metastasis; Transforming Growth Factor beta1

Astragali radix (Huang Qi, HQ), a well-known Chinese herbal medicine, is widely coadministered with many other drugs for treating diseases. The potential herb-drug interactions (HDIs) possibly occur during the combination therapy. P-glycoprotein (P-gp) and breast cancer resistance protein (BCRP) are the crucial targets that mediate the production of HDIs. We previously observed that HQ and its three main bioactive compounds, including Astragaloside IV (AS-IV), calycosin (CS) and formononetin (FMNT), could significantly induce the expression of P-gp and BCRP in HepG2 cells in vitro. However, their modulations on the function of P-gp and BCRP remain unknown; their impact on these two proteins expression in vivo is not clear; the exact regulatory mechanism has also not yet been explored.. This study aimed to investigate the impact of HQ, AS-IV, CS and FMNT on P-gp and BCRP in vivo, and the exact regulatory mechanism involved. The effects of HQ and these compounds on the function of P-gp and BCRP were also studied.. HQ, AS-IV, CS and FMNT significantly upregulated the P-gp and BCRP expression in the liver of wild-type mice. The induction was significantly reversed in the Nrf2. Our results proved that HQ and its main bioactive compounds could induce the P-gp and BCRP expression through the activation of the Nrf2-mediated signaling pathway. HQ and these compounds also significantly enhanced the efflux activity of P-gp and BCRP, and the increased intracellular ATP levels were likely involved in the increased P-gp and BCRP function. These results suggested that potentially HDIs likely occurred when HQ was used concomitantly with other drugs that are substrates of P-gp and BCRP.

Topics: Adenosine Triphosphate; Animals; Astragalus propinquus; ATP Binding Cassette Transporter, Subfamily B, Member 1; ATP Binding Cassette Transporter, Subfamily G, Member 2; Breast Neoplasms; Drugs, Chinese Herbal; Hep G2 Cells; Herb-Drug Interactions; Humans; Isoflavones; Liver; Mice, Inbred C57BL; Mice, Knockout; NF-E2-Related Factor 2; Saponins; Triterpenes

Previous studies report the upregulation of the secretory Rab27B small GTPase in human breast cancer, which could promote invasive growth and metastasis in estrogen receptor (ER)-positive breast cancer cells. However, there is limited evidence for its role in ER-negative breast cancer, along with the signaling pathways. Consistent with previous studies, we here confirmed that Rab27B is upregulated in breast tumor tissue in comparison with normal breast tissue. In addition, in ER-negative breast cancer cell line MDA-MB-231, when the levels of Rab27B expression were further elevated by transduction with recombinant lentivirus vector, migration and invasion assays demonstrated that cell migration and invasion was significantly stimulated. Moreover, Rab27B overexpression increased levels of β-catenin, followed by upregulation of vascular endothelial growth factor (VEGF). Our findings reveal a key function for the Rab27B-mediated modulation of β-catenin and VEGF in ER-negative breast cancer cell metastasis. Notably, the suppressed expression of Rab27B, β-catenin and VEGF was found in calycosin-treated MDA-MB-231 cells, accompanied with decreased invasive and migratory potential of these cells. What's more, these inhibitory effects of calycosin were all attenuated by Rab27B overexpression. The results demonstrated that calycosin-induced inhibition of migration and invasion in ER-negative breast cancer cells may be associated with the inactivation of Rab27B-dependent signaling, and suggest that antagonism of this pathway by calycosin may offer alternative therapeutic strategy for the aggressive breast cancer.

Topics: Breast Neoplasms; Cell Line, Tumor; Cell Movement; Down-Regulation; Female; Gene Expression Regulation, Neoplastic; Humans; Isoflavones; Neoplasm Invasiveness; rab GTP-Binding Proteins; Receptors, Estrogen; Signal Transduction; Tissue Array Analysis

Clinically, breast cancer is generally classified into estrogen receptor-positive (ER+) or estrogen receptor-negative (ER-) subtypes. The phytoestrogen calycosin has been shown to inhibit the proliferation of ER+ cells, which may be mediated by a feedback loop that involves miR-375, RAS dexamethasone-induced 1 (RASD1), and ERα. However, how calycosin acts on ER- breast cancer cells remains unclear.. Here, we show that calycosin inhibited the proliferation of both ER- (MDA-MB-468 and SKBR3) and ER+ breast cancer cells (MCF-7 and T47D) and that these inhibitory effects were associated with the up-regulation of the long non-coding RNA (lncRNA) WDR7-7. For the first time, we demonstrate that the expression of WDR7-7 is reduced in breast cancer cell lines and that the overexpression of WDR7-7 inhibits growth through a mechanism that involves G-protein coupled estrogen receptor 30 (GPR30). Meanwhile, we show that calycosin stimulated the WDR7-7-GPR30 signaling pathway in MCF-7, T47D, MDA-MB-468, and SKBR3 breast cancer cells. In contrast, in MCF10A and GPR30-deficient MDA-MB-231 cells, due to a lack of WDR7-7-GPR30 for activation, calycosin failed to inhibit cell growth. Additionally, in all four GPR30-positive breast cancer lines, calycosin decreased the phosphorylation levels of SRC, EGFR, ERK1/2 and Akt, but the inhibition of WDR7-7 blocked these changes and increased proliferation. In mice bearing MCF-7 or SKBR3 xenografts, tumor growth was inhibited by calycosin, and changes in expression the levels of WDR7-7 and GPR30 in tumor tissues were similar to those in cultured MCF-7 and SKBR3 cells.. These results suggest the possibility that calycosin inhibited the proliferation of breast cancer cells, at least partially, through WDR7-7-GPR30 signaling, which may explain why calycosin can exert inhibitory effects on ER- breast cancer.

Topics: Animals; Antineoplastic Agents, Phytogenic; Breast Neoplasms; Cell Line, Tumor; Cell Proliferation; Female; Gene Expression Regulation, Neoplastic; Humans; Isoflavones; MCF-7 Cells; Mice; Receptors, Estrogen; Receptors, G-Protein-Coupled; RNA, Long Noncoding; Signal Transduction; Up-Regulation; Xenograft Model Antitumor Assays

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.. 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.. 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.. 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.

Topics: Apoptosis; Breast Neoplasms; Cell Line, Tumor; Cell Movement; Cell Proliferation; Down-Regulation; Drugs, Chinese Herbal; Female; Forkhead Transcription Factors; Humans; Isoflavones; Matrix Metalloproteinase 9; MCF-7 Cells; Vascular Endothelial Growth Factor A

Danggui Buxue Tang (DBT), a Chinese herbal decoction containing Astragali Radix (AR; roots of Astragalus memebranaceus (Fisch.) Bunge var. mongholicus (Bunge) Hsiao) and Angelicae Sinensis Radix (ASR; roots of Angelica sinensis Oliv.) at a weight ratio of 5:1, is used to improve menopausal syndromes in women. Several lines of evidence indicate that DBT has strong estrogenic property; however, the action mechanism of this 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 key compound of DBT in responsible for such estrogenic properties.. We aimed to determine the role of calycosin in DBT in terms of its estrogenic functions by the creation of calycosin-depleted DBT (DBTΔcal) and calycosin-added DBT (DBT+cal) herbal extracts.. The signalings triggered by DBT∆cal, DBT+cal, and parental DBT were compared in cultured MCF-7 cells by determining: (i) the activation of estrogen responsive element; (ii) the phosphorylation of estrogen receptor α (ERα); and (iii) the phosphorylation of Erk1/2. The DBT-induced responses were in dose- and/or time-dependent manners.. The estrogenic signals triggered by DBT were markedly reduced in DBTΔcal, and in contrast the addition of calycosin in DBT, i.e. DBT+cal, enhanced the responses by 2-5 folds; however, calycosin alone did not show such properties. In parallel, the DBT-induced responses could be significantly blocked by inhibitors for estrogen receptor and mitogen activated protein kinases.. Thus, we hypothesize that calycosin is an indispensable chemical in DBT, and which plays a linker in orchestrating multi-components of DBT as to achieve the maximal estrogenic functions. These discoveries should be invaluable in drug development and in investigating the modernization of traditional Chinese medicine from a new perspective.

Topics: Breast Neoplasms; Dose-Response Relationship, Drug; Drugs, Chinese Herbal; Estradiol; Estrogen Receptor alpha; Estrogens; Female; HEK293 Cells; Humans; Isoflavones; MCF-7 Cells; Mitogen-Activated Protein Kinase 1; Mitogen-Activated Protein Kinase 3; Phosphorylation; Phytotherapy; Plants, Medicinal; Response Elements; Signal Transduction; Time Factors; Transfection

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

We previously reported that calycosin, a natural phytoestrogen structurally similar to estrogen, successfully triggered apoptosis of estrogen receptor (ER)-positive breast cancer cell line, MCF-7. To better understand the antitumor activities of calycosin against breast cancer, besides MCF-7 cells, another ER-positive cell line T-47D was analyzed here, with ER-negative cell lines (MDA-231, MDA-435) as control. Notably, calycosin led to inhibited cell proliferation and apoptosis only in ER-positive cells, particularly in MCF-7 cells, whereas no such effect was observed in ER-negative cells. Then we investigated whether regulation of ERβ, a subtype of ER, contributed to calycosin-induced apoptosis in breast cancer cells. The results showed that incubation of calycosin resulted in enhanced expression ERβ in MCF-7 and T-47D cells, rather than MDA-231 and MDA-435 cells. Moreover, with the upregulation of ERβ, successive changes in downstream signaling pathways were found, including inactivation of insulin-like growth factor 1 receptor (IGF-1R), then stimulation of p38 MAPK and suppression of the serine/threonine kinase (Akt), and finally poly(ADP-ribose) polymerase 1 (PARP-1) cleavage. However, the other two members of the mitogen-activated protein kinase (MAPK) family, extracellular signal-regulated kinase (ERK) 1/2 and c-Jun N-terminal kinase (JNK), were not consequently regulated by downregulated IGF-1R, indicating ERK 1/2 and JNK pathways were not necessary to allow proliferation inhibition by calycosin. Taken together, our results indicate that calycosin tends to inhibit growth and induce apoptosis in ER-positive breast cancer cells, which is mediated by ERβ-induced inhibition of IGF-1R, along with the selective regulation of MAPK and phosphatidylinositol 3-kinase (PI3K)/Akt pathways.

Topics: Breast Neoplasms; Cell Death; Cell Line, Tumor; Cell Proliferation; Dose-Response Relationship, Drug; Estrogen Receptor beta; Female; Gene Expression Regulation, Neoplastic; Humans; Isoflavones; MCF-7 Cells; p38 Mitogen-Activated Protein Kinases; Phosphatidylinositol 3-Kinases; Poly (ADP-Ribose) Polymerase-1; Poly(ADP-ribose) Polymerases; Proto-Oncogene Proteins c-akt; Receptor, IGF Type 1; Signal Transduction

Breast cancer is a leading cause of cancer death among women, and the failure of normal apoptosis has been proved in the development of breast cancer. The phytoestrogen, calycosin, is extracted from Chinese medical herb Radix astragali. We recently reported that calycosin successfully stimulated proliferation of ER-positive MCF-7 human breast cancer cells at low concentration. In the present study, we assessed the proapoptotic function of calycosin in MCF-7 cells at high concentration in vitro, as well as the possible mechanism of its effect. MCF-7 cells were treated with different concentrations of calycosin, and then detected by MTT assay for cellular viability, Hoechst assay, and flow cytometry for apoptosis. RASD1 is identified as a Ras-family member and a regulator in MAPK-mediated cascade leading to cell proliferation or apoptosis. To provide insight into the functions of RASD1 signaling pathway in calycosin-induced apoptosis, the expression of Bcl-2, Bax, and RASD1 in calycosin-treated cells were determined by Western blot assay. The results showed that high concentrations of calycosin significantly suppressed the proliferation of MCF-7 cells and promoted cell apoptosis. Moreover, compared with control group, the expression of Bcl-2 decreased with calycosin in MCF-7 cells, while Bax increased, which was significantly correlated with elevated expression of RASD1. Together, we present evidence that at relatively high concentration calycosin triggered cell apoptosis through the mitochondrial apoptotic pathway by upregulating RASD1. And for the first time, this study revealed that calycosin may have potential as a therapeutic agent for the treatment of breast cancer.

Topics: Apoptosis; bcl-2-Associated X Protein; Breast Neoplasms; Cell Line, Tumor; Cell Proliferation; Female; Humans; Isoflavones; Mitochondria; Proto-Oncogene Proteins c-bcl-2; ras Proteins; Signal Transduction; Up-Regulation

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.. 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.. 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.. 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.

Topics: Antineoplastic Agents, Phytogenic; Apoptosis; Breast Neoplasms; Cell Line, Tumor; Cell Proliferation; Down-Regulation; Estrogen Receptor beta; Female; Humans; Isoflavones; MCF-7 Cells; MicroRNAs; Poly (ADP-Ribose) Polymerase-1; Poly(ADP-ribose) Polymerases; Receptor, IGF Type 1; RNA, Messenger; Up-Regulation

Calycosin is a main active component of the herb Radix Astragali, and is considered as a phytoestrogen. Its effects in vivo may be either estrogenic or antiestrogenic, mainly depending upon the estrogen levels. This study was a continuation of our investigations of calycosin's promotion of the proliferation of estrogen receptor (ER)-positive cells via ERs and ERK1/2 activation in vitro and in vivo. ER-positive MCF-7 (human breast cancer) cells were treated with different concentrations of calycosin. Proliferation of the cells treated with calycosin was assayed by CCK8. Apoptosis in the treated cells was measured by flow cytometry. The protein expression of ERK1/2 in treated cells was determined by Western blot. In addition, the in vivo expression of ERα in the uterine tissues of ovariectomized (OVX) mice was assessed by immunohistochemistry. Compared with the control, low concentrations of calycosin (2-8 μM) stimulated the proliferation of MCF-7 cells and decreased the percentage of early apoptosis. The level of p-ERK1/2 was also downregulated at these low concentrations. Furthermore, we found that an ERK1/2 inhibitor significantly blocked the effect of calycosin in MCF-7 cells. In the in vivo studies, calycosin stimulated a dramatic increase in uterine weight and downregulated the level of ERα protein in OVX mice. This study demonstrated that at relatively low concentrations calycosin had stimulatory effects on the proliferation of MCF-7 cells, and we conclude that this is due to its estrogenic effect.

Topics: Animals; Apoptosis; Blotting, Western; Breast Neoplasms; Cell Line, Tumor; Cell Proliferation; Estrogen Receptor alpha; Estrogens; Female; Flow Cytometry; Gene Expression Regulation; Humans; Immunohistochemistry; Isoflavones; Mice; Mitogen-Activated Protein Kinase 1; Mitogen-Activated Protein Kinase 3; Molecular Structure

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