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

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

BACKGROUND The aim of our study was to elucidate the biological targets and pharmacological mechanisms for calycosin (CC) against colorectal cancer (CRC) through an approach of system pharmacology. MATERIAL AND METHODS Using a web-based platform, all CRC-causing genes were identified using a database of gene-disease associations (DisGeNET), and all well-known genes of CC identified using the databases of prediction of protein targets of small molecules (Swiss Target Prediction), drug classification, and target prediction (SuperPred). The carefully selected genes of CRC and CC were concurrently constructed by using a database of functional protein association networks (STRING), and use of software for visualizing complex networks (Cytoscape), characterized with production of protein-protein interaction (PPI) network of CC against CRC. The important biological targets of CC against CRC were identified through topological analysis, then the biological processes and molecular pathways of CC against CRC were further revealed for testing these important biotargets by enrichment assays. RESULTS We found that the key predictive targets of CC against CRC were estrogen receptor 2 (ESR2), ATP-binding cassette sub-family G member 2 (ABCG2), breast cancer type 1 susceptibility protein (BRCA1), estrogen receptor 1 (ESR1), cytochrome p450 19A1 (CYP19A1), and epidermal growth factor receptor (EGFR). Visual analysis revealed that the biological processes of CC against CRC were positively linked to hormonal metabolism, regulation of genes, transport, cell communication, and signal transduction. Further, the interrelated molecular pathways were chiefly related to endogenous nuclear estrogen receptor alpha network, forkhead box protein A1 (FOXA1) transcription factor network, activating transcription factor 2 (ATF2) transcription factor network, regulation of telomerase, plasma membrane estrogen receptor signaling, estrogen biosynthesis, androgen receptor, FOXA transcription factor networks, estrogen biosynthesis, and phosphorylation of repair proteins. CONCLUSIONS Use of system pharmacology revealed the biotargets, biological processes, and pharmacological pathways of CC against CRC. Intriguingly, the identifiable predictive biomolecules are likely potential targets for effectively treating CRC.

Topics: Aromatase; ATP Binding Cassette Transporter, Subfamily G, Member 2; Biomarkers, Tumor; BRCA1 Protein; Colonic Neoplasms; Colorectal Neoplasms; Computational Biology; Databases, Genetic; ErbB Receptors; Estrogen Receptor alpha; Estrogen Receptor beta; Gene Expression Profiling; Gene Expression Regulation, Neoplastic; Gene Regulatory Networks; Isoflavones; Neoplasm Proteins; Pharmacological and Toxicological Phenomena; Protein Interaction Maps; Signal Transduction; Systems Analysis

Calycosin, the main component extractable from the herb Radix astragali, has been shown to inhibit cellular proliferation and induce apoptosis in several cancer cell lines, but the underlying mechanisms by the way in which this occurs remain unclear. In the present study, we aimed to determine the potential effects of calycosin on proliferation in colorectal cancer cells in vitro and in vivo and to elucidate the underlying molecular mechanisms of action. Colorectal cancer cell lines SW480 and LoVo and cervical cancer cell line HeLa were treated with various concentrations of calycosin or plus ER beta (ERβ) inhibitor PHTPP. The CCK8 assay, flow cytometry, and Hoechst 33258 stain were used to assess the effects on cellular proliferation and apoptosis. The mRNA levels of ERβ and miR-95 were quantified by real-time PCR. The protein expression levels of ERβ, ERα, IGF-1R, and p-Akt were evaluated by Western blot analysis. We demonstrated that calycosin inhibited the proliferation in SW480 and LoVo cells and induced apoptosis, particularly in SW480 cells, but not in HeLa cells. Calycosin increased ERβ expression and reduced the ERα, IGF-1R, and p-Akt expression alongside down-regulation of miR-95 in SW480 cells. Inhibiting ERβ blocked the change of miR-95 and the resulting increase in apoptosis in SW480 cells. Additionally, calycosin significantly suppressed xenograft tumor growth in nude mice. In conclusion, calycosin exerts an inhibitory effect on proliferation of CRC cells in vivo and in vitro, through ERβ-mediated regulation of the IGF-1R, PI3K/Akt signaling pathways and of miR-95 expression.

Topics: Animals; Apoptosis; Cell Line, Tumor; Cell Proliferation; Cell Shape; Colorectal Neoplasms; Estrogen Receptor beta; Female; Gene Expression Regulation, Neoplastic; Humans; Isoflavones; Mice; MicroRNAs; Phosphatidylinositol 3-Kinases; Proto-Oncogene Proteins c-akt; Receptor, IGF Type 1; RNA, Messenger; Signal Transduction

Prior studies have suggested that a high intake of isoflavonoids is associated with a protective effect against hormone-related cancers, such as colorectal cancer (CRC). Calycosin, a main component of isoflavones, has been shown to suppress the growth of hormone-dependent tumors through an ERβ-mediated signaling pathway. However, the effects of calycosin on CRC remain unclear. In this study, we aimed to investigate the anti-tumor activities of calycosin on CRC and its potential mechanism. HCT-116 cells were treated with calycosin. Cell proliferation, apoptosis and invasiveness were measured by MTT assay, flow cytometry and transwell invasion assay, respectively. mRNA levels of ER beta (ERβ) and miR-17 were quantified by real-time PCR. Protein expressions of ERβ and phosphatase and tensin homolog deleted on chromosome ten (PTEN) were determined by western blotting. We found that calycosin significantly induced apoptosis, and inhibited proliferation and invasiveness of HCT-116 cells in a dose-dependent manner. In addition, ERβ expression significantly increased in calycosin-treated HCT-116 cells, followed by a decrease of miR-17, and up-regulation of PTEN. Our results indicate that calycosin has an inhibitory effect on CRC, which might be obtained by ERβ-mediated regulation of miR-17 and PTEN expression.

Topics: Apoptosis; Cell Proliferation; Colorectal Neoplasms; Estrogen Receptor beta; Gene Expression Regulation, Neoplastic; HCT116 Cells; Humans; Isoflavones; MicroRNAs; PTEN Phosphohydrolase; Signal Transduction