PMID: 

J Cell Physiol. 2019 Aug ;234(10):17305-17313. Epub 2019 Feb 20. PMID: 30790283

Abstract Title: 

In vitro and in vivo studies of antiosteosarcoma activities of formononetin.

Abstract: 

Osteogenic sarcoma (OGS) is a primary bone cancer, characterized by aggressive neoplasm from mesenchymal oncogenesis. However, the clinical therapeutic regimen against OGS is limited. Therefore, potential medication warrants to be further developed. Our previous study indicates that formononetin (FN) exerts effective pharmacological activity against OGS. This study aimed to further decipher the molecular mechanism behind this benefit. Patients with OGS were recruited for clinical data assay and immunoassay. Human OGS cell line (U2OS) and tumor-bearing nude mice were subjected to a battery of biochemical analyses and immunoassays for integrative evaluation of FN-exerted anti-OGS effects. In human data, OGS samples showed increased expressions of ERα, p-PI3KCA, and p-AKTproteins, followed by notably upregulated miR-375 content in comparison with that in OGS-free. In addition, FN-treated U2OS cells showed inhibited cell proliferation, elevated lactic dehydrogenase production and lowered endogenous miR-375 level in cells. Further, reduced immunopositive cells of Ki-67, p-PI3KCA, and p-AKTwere observed by the treatments of FN, while the intracellular Bax- and Apaf-1-positive cells were increased dose-dependently. Beneficially, FN-treated tumor-bearing mice exhibited reduced tumor mass and intercellular miR-375 expression. Meanwhile, immuno-labeled cells and proteins of Bax, Caspase-3, and Apaf-1 in FN-treated mice were increased dose-dependently, whereas ERα, p-PI3KCA, and p-AKTpositive cells and proteins were downregulated, respectively. Collectively, our current results elucidate that FN exerts effective therapeutic benefits against OGS, and the pharmacological mechanism may be related to promoting cell apoptosis by inactivating intracellular miR-375/ERα-PI3K/AKT cascaded pathway.

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PMID: 

J Cell Mol Med. 2019 May ;23(5):3505-3511. Epub 2019 Mar 15. PMID: 30873755

Abstract Title: 

Anti-cancer targets of formononetin and molecular mechanisms in osteosarcoma: Findings of bioinformatic and experimental assays.

Abstract: 

In current study, a bioinformatic-based network pharmacology was used to identify the osteosarcoma (OGS)-pathological targets and formononetin (FN)-treated targets before the main core predictive biotargets were screened. In addition, all core targets were selected through a number of bioinformatic databases, followed by identification of predominant biological processes and signalling pathways of FN anti-OGS. Further, top three core targets of FN anti-OGS were determined as oestrogen receptor 1 (ESR1), tumour protein p53 (TP53), receptor tyrosine-protein kinase erbB-2 (ERBB2) respectively. In clinical biochemical data, the plasma samples of OGS showed the increased trends of alkaline phosphatase, triglyceride, blood glucose, lactate dehydrogenase, high-sensitive C-reactive protein and some immune cell counts when referenced to medical criteria. In clinicopathological examination, histological OGS sections resulted in increased positive cell counts of neoplastic ESR1, TP53, ERBB2. To further validate these corn proteins in experimental study in vivo, FN-treated tumour-bearing nude mice showed intracellular reductions of ESR1, TP53, ERBB2 positive expressions, accompanied with visibly reduced tumour weights. Collectively, our bioinformatic and experimental findings disclosed main core targets, biological processes and signalling pathways of FN anti-OGS. Interestingly, the top core targets were representatively validated following FN treatment in vivo. Therefore, we reasoned that these predictive targets might be the potential biomarkers for screening and treating osteosarcoma.

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PMID: 

Mol Brain. 2019 04 8 ;12(1):36. Epub 2019 Apr 8. PMID: 30961625

Abstract Title: 

Anxiolytic effects of Formononetin in an inflammatory pain mouse model.

Abstract: 

Chronic pain is commonly accompanied with anxiety disorder, which complicates treatment. In this study, we investigated the analgesic and anxiolytic effects of Formononetin (FMNT), an active component of traditional Chinese medicine red clover (Trifolium pratense L.) that is capable of protecting neurons from N-methyl-D-aspartate (NMDA)-evoked excitotoxic injury, on mice suffering from complete Freund's adjuvant (CFA)-induced chronic inflammatory pain. The results show that FMNT administration significantly reduces anxiety-like behavior but does not affect the nociceptive threshold in CFA-injected mice. The treatment reverses the upregulation of NMDA, GluA1, and GABAreceptors, as well as PSD95 and CREB in the basolateral amygdala (BLA). The effects of FMNT on NMDA receptors and CREB binding protein (CBP) were further confirmed by the potential structure combination between these compounds, which was analyzed by in silico docking technology. FMNT also inhibits the activation of the NF-κB signaling pathway and microglia in the BLA of mice suffering from chronic inflammatory pain. Therefore, the anxiolytic effects of FMNT are partially due to the attenuation of inflammation and neuronal hyperexcitability through the inhibition of NMDA receptor and CBP in the BLA.

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PMID: 

CNS Neurol Disord Drug Targets. 2019 Aug 7. Epub 2019 Aug 7. PMID: 31389320

Abstract Title: 

Formononetin ameliorates cognitive disorder via PGC-1α pathway in neuroinflammation conditions in high-fat diet-induced mice.

Abstract: 

Alzheimer's disease (AD) is one of the most common neurodegenerative diseases in many modern societies. Previous studies have shown that neuroinflammation is one of the pathogenesis of AD. Formononetin (FN), an isoflavone compound extracted from Trifolium pratense L., has been found to have various properties including anti-obesity, anti-inflammation, and neuroprotective effects. The present study focused on the protective activities of FN on a high-fat diet (HFD)-induced cognitive decline and explored the underlying mechanisms. We found that FN (20, 40 mg/kg) significantly attenuated the learning and memory deficits companied by weight improvement and decreased the levels of blood glucose, total cholesterol (TC) and triglyceride (TG) in HFD-induced mice. Meanwhile, we observed HFD significantly caused the Tau hyperphosphorylation in the hippocampus of mice, whereas FN reversed this effect. Additionally, FN markedly reduced the levels of inflammation cytokines IL-1β and TNF-α in HFD-induced mice. The mechanism study showed that FN suppressed the pro-inflammatory NF-κB signaling and enhanced the anti-inflammatory Nrf-2/HO-1 signaling, which might be related to the regulation of PGC-1α in the hippocampus of HFD-induced mice. Taken together, our results showed that FN could improve the cognitive function by inhibiting neuroinflammation, which is attributed to the regulation of PGC-1α pathway in HFD-induced mice.

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PMID: 

Biomolecules. 2019 Jul 7 ;9(7). Epub 2019 Jul 7. PMID: 31284669

Abstract Title: 

Formononetin Regulates Multiple Oncogenic Signaling Cascades and Enhances Sensitivity to Bortezomib in a Multiple Myeloma Mouse Model.

Abstract: 

Here, we determined the anti-neoplastic actions of formononetin (FT) against multiple myeloma (MM) and elucidated its possible mode of action. It was observed that FT enhanced the apoptosis caused by bortezomib (Bor) and mitigated proliferation in MM cells, and these events are regulated by nuclear factor-κB (NF-κB), phosphatidylinositol 3-kinase (PI3K)/AKT, and activator protein-1 (AP-1) activation. We further noted that FT treatment reduced the levels of diverse tumorigenic proteins involved in myeloma progression and survival. Interestingly, we observed that FT also blocked persistent NF-κB, PI3K/AKT, and AP-1 activation in myeloma cells. FT suppressed the activation of these oncogenic cascades by affecting a number of signaling molecules involved in their cellular regulation. In addition, FT augmented tumor growth-inhibitory potential of Bor in MM preclinical mouse model. Thus, FT can beemployed with proteasomal inhibitors for myeloma therapy by regulating the activation of diverse oncogenic transcription factors involved in myeloma growth.

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PMID: 

Front Pharmacol. 2019 ;10:820. Epub 2019 Jul 26. PMID: 31402861

Abstract Title: 

Formononetin: A Review of Its Anticancer Potentials and Mechanisms.

Abstract: 

Cancer, a complex yet common disease, is caused by uncontrolled cell division and abnormal cell growth due to a variety of gene mutations. Seeking effective treatments for cancer is a major research focus, as the incidence of cancer is on the rise and drug resistance to existing anti-cancer drugs is major concern. Natural products have the potential to yield unique molecules and combinations of substances that may be effective against cancer with relatively low toxicity/better side effect profile compared to standard anticancer therapy. Drug discovery work with natural products has demonstrated that natural compounds display a wide range of biological activities correlating to anticancer effects. In this review, we discuss formononetin (CHO), which originates mainly from red clovers and the Chinese herb. The compound comes from a class of 7-hydroisoflavones with a substitution of methoxy group at position 4. Formononetin elicits antitumorigenic propertiesandby modulating numerous signaling pathways to induce cell apoptosis (by intrinsic pathway involving Bax, Bcl-2, and caspase-3 proteins) and cell cycle arrest (by regulating mediators like cyclin A, cyclin B1, and cyclin D1), suppress cell proliferation [by signal transducer and activator of transcription (STAT) activation, phosphatidylinositol 3-kinase/protein kinase-B (PI3K/AKT), and mitogen-activated protein kinase (MAPK) signaling pathway], and inhibit cell invasion [by regulating growth factors vascular endothelial growth factor (VEGF) and Fibroblast growth factor 2 (FGF2), and matrix metalloproteinase (MMP)-2 and MMP-9 proteins]. Co-treatment with other chemotherapy drugs such as bortezomib, LY2940002, U0126, sunitinib, epirubicin, doxorubicin, temozolomide, and metformin enhances the anticancer potential of both formononetin and the respective drugs through synergistic effect. Compiling the evidence thus far highlights the potential of formononetin to be a promising candidate for chemoprevention and chemotherapy.

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PMID: 

Eur J Pharmacol. 2019 Oct 15 ;861:172619. Epub 2019 Aug 17. PMID: 31430458

Abstract Title: 

Medical findings of nasopharyngeal carcinoma patients and anti-tumor benefits of formononetin.

Abstract: 

Epidemiologically, a malignancy of nasopharyngeal carcinoma (NPC) is endemic in worldwide, characterized with high invasiveness and lethality. Formononetin (FN), an anti-tumor bioactive component, is found to exert anti-proliferative activity against NPC cells. However, the invasive pharmacological activities of FN against NPC have not yet been investigated. In this study, the human NPC data and samples were used for further assays. In addition, a human cell line of CNE2 was used to evaluate the anti-invasive effects of formononetin and the underlying mechanism. As results, advanced NPC patients were diagnosed through validation of blood tumor markers and medical images, and the results showed increased Lamin A/C and cytokeratin 19 (CK19) expressions in carcinomatous sections. In experimental study in vitro, FN-treated CNE2 cells exhibited inhibited cellular proliferation, promoted cell apoptosis, and decreased would healing process, reduced cellular migratory capability, respectively. Furthermore, FN-treated CNE2 cells resulted in down-regulated expressions of b-cell lymphoma-2 (Bcl-2), extracellular regulated protein kinases1/2 (ERK1/2), Lamin A/C and CK19 in a dose-dependent manner, while intracellular Bax expression was elevated. Taken together, these clinical findings elucidate invasive characteristics of human NPC samples. Further, the anti-proliferative and invasive benefits of FN were achieved through suppression of cellular ERK1/2 pathway and inactivation of intracellular Lamin A/C signaling in NPC cells.

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PMID: 

Oncol Lett. 2019 Sep ;18(3):2248-2253. Epub 2019 Jul 5. PMID: 31452725

Abstract Title: 

Effects and significance of formononetin on expression levels of HIF-1α and VEGF in mouse cervical cancer tissue.

Abstract: 

Effects and significance of formononetin on the expression levels of hypoxia-inducible factor-1α (HIF-1α) and vascular endothelial growth factor (VEGF) in mouse cervical cancer tissue were investigated. The animal models of Balb/c nude mice with cervical cancer were established by the inoculation of HeLa cells, and randomly divided into positive control (n=10), cisplatin (n=15) and formononetin group (n=15). Mice were all sacrificed on the 31st day after administration, and their tumors were excised and weighed to calculate tumor inhibition rate. At the same time, their cancer tissues were obtained. RT-qPCR was used for detecting the mRNA expression levels of HIF-1α and VEGF, and western blotting for detecting the protein expression levels. During the medication intervention, mice in the formononetin group had no obvious adverse reactions, and were in good condition, whereas mice in the cisplatin group had poor appetite, drooping spirits and decreased activity. Mice in the cisplatin and the formononetin groups had significantly lower tumor mass and volume than those in the positive control group (P

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PMID: 

J Nutr Biochem. 2019 Jul 25 ;73:108214. Epub 2019 Jul 25. PMID: 31520816

Abstract Title: 

Formononetin alleviates hepatic steatosis by facilitating TFEB-mediated lysosome biogenesis and lipophagy.

Abstract: 

Formononetin has been reported to ameliorate hyperlipidemia and obesity, but its effect and mechanism of action in anti-non-alcoholic fatty liver disease (NAFLD) remain unclear. Lipophagy is a critical protective mechanism during steatosis development that results in the decomposition of lipid droplets through autophagy and the prevention of cellular lipid accumulation. This study aimed to investigate the beneficial role of formononetin in treating NAFLD and explore the mechanism of lipophagy in formononetin anti-hepatic steatosis effects. Formononetin treatment significantly ameliorated hepatic steatosis in HFD mice. Consistently, formononetin also reduced FFAs-stimulated lipid accumulation in HepG2 cells and primary mouse hepatocytes. Further analysis revealed that steatosis increased LC3B-II, a marker of autophagy, but caused blockade of autophagic flux associated with a lack of lysosomes. Treatment with formononetin promoted lysosome biogenesis and autophagosome-lysosome fusion, relieving the blockade in autophagic flux and further induced lipophagy. Mechanistically, formononetin activated adenosine monophosphate activated protein kinase (AMPK) and promoted subsequent nuclear translocation of transcription factor EB (TFEB), a key regulator of lysosome biogenesis. TFEB inhibition markedly abolished formononetin-induced lysosome biogenesis, autophagosome-lysosome fusion and lipophagy and concomitantly alleviated lipid accumulation. Formononetin improved hepatic steatosis via TFEB-mediated lysosome biogenesis, which provides new evidence regarding formononetin's anti-NAFLD effects.

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PMID: 

Mol Med Rep. 2019 Sep ;20(3):2893-2901. Epub 2019 Jul 19. PMID: 31524234

Abstract Title: 

Formononetin ameliorates high glucose‑induced endothelial dysfunction by inhibiting the JAK/STAT signaling pathway.

Abstract: 

High glucose‑induced endothelial Janus kinase (JAK)/signal transducer and activator of transcription (STAT) signaling is associated with the development and progression of the vascular complications of diabetes. The present study aimed to investigate whether formononetin, a biologically active compound isolated from Astragalus membranaceus (Fisch.) Bge, was able to regulate the JAK/STAT signaling pathway, improving endothelial function. In the present study, formononetin was identified to act as a JAK2 inhibitor, similarly to tyrphostin AG 490 (AG490), by significantly inhibiting the phosphorylation and the mRNA expression levels of JAK2 and STAT in HUVECs exposed to high glucose levels. In addition, formononetin and AG490 improved the viability of HUVECs and inhibited the protein expression levels of caspase‑3. Furthermore, formononetin and AG490 attenuated the inflammatory response in HUVECsby downregulating the protein and mRNA expression levels of interleukin (IL)‑1β and intercellular adhesion molecule 1 (ICAM‑1). Formononetin and AG490 also restored nitric oxide (NO) synthesis in HUVECs. Notably, formononetin was able to reverse the abnormal levels of phosphorylated (p)‑JAK2, p‑STAT3, IL‑1β, ICAM‑1 and NO induced by cotreatment with high glucose and IL‑6, an agonist of the JAK/STAT signaling pathway. Additionally, the present results suggested that formononetin restored phenylephrine‑mediated contraction and acetylcholine‑induced relaxation in aortic tissues of rats fed a high‑glucose diet, in a dose‑dependent manner. Collectively, formononetin could improve endothelial function under glucose stress in vivo and in vitro, suggesting that formononetin may represent a novel potential therapeutic compound to treat diabetic vascular complications.

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