The angiogenesis-modulating effects of coumarin-derivatives.

Coumarin is a natural compound that is rich in plants. Coumarin and its derivates were reported to have many biological activities, such as anti-bacterial, anti-tumor, and anti-coagulation. In this study, we examined the angiogenic modulating activities of six previously synthesized coumarin derivatives (Compound #1-#6) in zebrafish embryos and further confirmed them in a chick model. According to the survival rate in a zebrafish model, Compound #1 (100 %), #2 (82.5-100 %), and #4 (100 %) showed much less toxicity than Compound #3 (19.2-100 %), #5 (0-100 %), and #6 (0-100 %). Using a green blood vessel fluorescent transgenic fish Tg(fli1:egfp) to record the angiogenesis-modulating effects of Compound #1, #2, and #4, we found that Compound #2 had the highest effects in interfering intersegmental vessel growth, subintestinal vein growth, and caudal vein plexus remodeling. Chick chorioallantoic membrane (CAM) assay also showed that Compound #2 exposure led to a reduction of blood vessel growth. Real-time PCR experiments revealed that Compound #2 significantly changed the expression of vascular growth-related genes flt1, cdh5, and nrp1a in zebrafish. Based on our data from zebrafish and chick models, a new coumarin-derivative (Compound #2) possesses anti-angiogenic activity with low toxicity, but further investigation in mammal models is asked to confirm our findings.

Coumarin Derivatives Inhibit ADP-Induced Platelet Activation and Aggregation.

Coumarin was first discovered in Tonka bean and then widely in other plants. Coumarin has an anticoagulant effect, and its derivative, warfarin, is a vitamin K analogue that inhibits the synthesis of clotting factors and is more widely used in the clinical treatment of endovascular embolism. At present, many artificial chemical synthesis methods can be used to modify the structure of coumarin to develop many effective drugs with low toxicity. In this study, we investigated the effects of six coumarin derivatives on the platelet aggregation induced by adenosine diphosphate (ADP). We found that the six coumarin derivatives inhibited the active form of GPIIb/IIIa on platelets and hence inhibit platelet aggregation. We found that 7-hydroxy-3-phenyl 4H-chromen-4-one (7-hydroxyflavone) had the most severe effect. In addition, we further analyzed the downstream signal transduction of the ADP receptor, including the release of calcium ions and the regulation of cAMP, which were inhibited by the six coumarin derivatives selected in this study. These results suggest that coumarin derivatives inhibit coagulation by inhibiting the synthesis of coagulation factors and they may also inhibit platelet aggregation.

Targeting HR Repair as a Synthetic Lethal Approach to Increase DNA Damage Sensitivity by a RAD52 Inhibitor in BRCA2-Deficient Cancer Cells.

BRCA mutation, one of the most common types of mutations in breast and ovarian cancer, has been suggested to be synthetically lethal with depletion of RAD52. Pharmacologically inhibiting RAD52 specifically eradicates BRCA-deficient cancer cells. In this study, we demonstrated that curcumin, a plant polyphenol, sensitizes BRCA2-deficient cells to CPT-11 by impairing RAD52 recombinase in MCF7 cells. More specifically, in MCF7-siBRCA2 cells, curcumin reduced homologous recombination, resulting in tumor growth suppression. Furthermore, a BRCA2-deficient cell line, Capan1, became resistant to CPT-11 when BRCA2 was reintroduced. In vivo, xenograft model studies showed that curcumin combined with CPT-11 reduced the growth of BRCA2-knockout MCF7 tumors but not MCF7 tumors. In conclusion, our data indicate that curcumin, which has RAD52 inhibitor activity, is a promising candidate for sensitizing BRCA2-deficient cells to DNA damage-based cancer therapies.

An octimibate derivative, Oxa17, enhances cholesterol efflux and exerts anti-inflammatory and atheroprotective effects in experimental atherosclerosis.

Atherosclerotic cardiovascular diseases (ASCVDs), associated with vascular inflammation and lipid dysregulation, are responsible for high morbidity and mortality rates globally. For ASCVD treatment, cholesterol efflux plays an atheroprotective role in ameliorating inflammation and lipid dysregulation. To develop a multidisciplinary agent for promoting cholesterol efflux, octimibate derivatives were screened and investigated for the expression of ATP-binding cassette transporter A1 (ABCA1). Western blotting and qPCR analysis were conducted to determine the molecular mechanism associated with ABCA1 expression in THP-1 macrophages; results revealed that Oxa17, an octimibate derivative, enhanced ABCA1 expression through liver X receptors alpha (LXRα) activation but not through the microRNA pathway. We also investigated the role of Oxa17 in high-fat diet (HFD)-fed mice used as an in vivo atherosclerosis-prone model. In ldlr-/- mice, Oxa17 increased plasma high-density lipoprotein (HDL) and reduced plaque formation in the aorta. Plaque stability improved via reduction of macrophage accumulation and via narrowing of the necrotic core size under Oxa17 treatment. Our study demonstrates that Oxa17 is a novel and potential agent for ASCVD treatment with atheroprotective and anti-inflammatory properties.

A chalcone derivative, 1m-6, exhibits atheroprotective effects by increasing cholesterol efflux and reducing inflammation-induced endothelial dysfunction.

BACKGROUND AND PURPOSE: Atherosclerosis, resulting from lipid dysregulation and vascular inflammation, causes atherosclerotic cardiovascular disease (ASCVD), which contributes to morbidity and mortality worldwide. Chalcone and its derivatives possess beneficial properties, including anti-inflammatory, antioxidant and antitumour activity with unknown cardioprotective effects. We aimed to develop an effective chalcone derivative with antiatherogenic potential. EXPERIMENTAL APPROACH: Human THP-1 cells and HUVECs were used as in vitro models. Western blots and real-time PCRs were performed to quantify protein, mRNA and miRNA expressions. The cholesterol efflux capacity was assayed by 3 H labelling of cholesterol. LDL receptor knockout (Ldlr-/- ) mice fed a high-fat diet were used as an in vivo atherogenesis model. Haematoxylin and eosin and oil red O staining were used to analyse plaque formation. KEY RESULTS: Using ATP-binding cassette transporter A1 (ABCA1) expression we identified the chalcone derivative, 1m-6, which enhances ABCA1 expression and promotes cholesterol efflux in THP-1 macrophages. Moreover, 1m-6 stabilizes ABCA1 mRNA and suppresses the expression of potential ABCA1-regulating miRNAs through nuclear factor erythroid 2-related factor 2 (Nrf2)/haem oxygenase-1 (HO-1) signalling. Additionally, 1m-6 significantly inhibits TNF-α-induced expression of adhesion molecules, vascular cell adhesion molecule 1 (VCAM-1) and intercellular adhesion molecule 1 (ICAM-1), plus production of proinflammatory cytokines via inhibition of JAK/STAT3 activation and the modulation of Nrf2/HO-1 signalling in HUVECs. In atherosclerosis-prone mice, 1m-6 significantly reduces lipid accumulation and atherosclerotic plaque formation. CONCLUSION AND IMPLICATIONS: Our study demonstrates that 1m-6 produces promising atheroprotective effects by enhancing cholesterol efflux and suppressing inflammation-induced endothelial dysfunction, which opens a new avenue for treating ASCVD. LINKED ARTICLES: This article is part of a themed issue on Risk factors, comorbidities, and comedications in cardioprotection. To view the other articles in this section visit http://onlinelibrary.wiley.com/doi/10.1111/bph.v177.23/issuetoc.

Discovery of T-1101 tosylate as a first-in-class clinical candidate for Hec1/Nek2 inhibition in cancer therapy.

Highly expressed in cancer 1 (Hec1) plays an essential role in mitosis and is correlated with cancer formation, progression, and survival. Phosphorylation of Hec1 by Nek2 kinase is essential for its mitotic function, thus any disruption of Hec1/Nek2 protein-protein interaction has potential for cancer therapy. We have developed T-1101 tosylate (9j tosylate, 9j formerly known as TAI-95), optimized from 4-aryl-N-pyridinylcarbonyl-2-aminothiazole of scaffold 9 by introducing various C-4' substituents to enhance potency and water solubility, as a first-in-class oral clinical candidate for Hec1 inhibition with potential for cancer therapy. T-1101 has good oral absorption, along with potent in vitro antiproliferative activity (IC50: 14.8-21.5 nM). It can achieve high concentrations in Huh-7 and MDA-MB-231 tumor tissues, and showed promise in antitumor activity in mice bearing human tumor xenografts of liver cancer (Huh-7), as well as of breast cancer (BT474, MDA-MB-231, and MCF7) with oral administration. Oral co-administration of T-1101 halved the dose of sorafenib (25 mg/kg to 12.5 mg/kg) required to exhibit comparable in vivo activity towards Huh-7 xenografts. Cellular events resulting from Hec1/Nek2 inhibition with T-1101 treatment include Nek2 degradation, chromosomal misalignment, and apoptotic cell death. A combination of T-1101 with either of doxorubicin, paclitaxel, and topotecan in select cancer cells also resulted in synergistic effects. Inactivity of T-1101 on non-cancerous cells, a panel of kinases, and hERG demonstrates cancer specificity, target specificity, and cardiac safety, respectively. Subsequent salt screening showed that T-1101 tosylate has good oral AUC (62.5 µM·h), bioavailability (F = 77.4%), and thermal stability. T-1101 tosylate is currently in phase I clinical trials as an orally administered drug for cancer therapy.

Chalcone Derivatives Enhance ATP-Binding Cassette Transporters A1 in Human THP-1 Macrophages.

Atherosclerosis is a process of imbalanced lipid metabolism in the vascular walls. The underlying pathology mainly involves the deposition of oxidized lipids in the endothelium and the accumulation of cholesterol in macrophages. Macrophages export excessive cholesterol (cholesterol efflux) through ATP-binding cassette transporter A1 (ABCA1) to counter the progression of atherosclerosis. We synthesized novel chalcone derivatives and assessed their effects and the underlying mechanisms on ABCA1 expression in macrophages. Human THP-1 macrophages were treated with synthetic chalcone derivatives for 24 h. In Western blot and flow cytometry analyses, a chalcone derivative, (E)-1-(3,4-diisopropoxyphenyl)-3-(4-isopropoxy-3-methoxyphenyl)prop- 2-en-1-one (1m), was observed to significantly enhance ABCA1 protein expression in THP-1 cells (10 µM, 24 h). Levels of mRNA of ABCA1 and liver X receptor alpha (LXRα) were quantified using a real-time quantitative polymerase chain reaction technique and were found to be significantly increased after treatment with the novel chalcone derivative 1m. Several microRNAs, including miR155, miR758, miR10b, miR145, miR33, and miR106b, which functionally inhibit ABCA1 expression were suppressed after treatment with 1m. Collectively, 1m increases ABCA1 expression in human THP-1 macrophages. The mechanisms involve the activation of the LXRα-ABCA1 pathway and suppression of certain microRNAs that regulate ABCA1 expression.

Pro-Angiogenic Effects of Chalcone Derivatives in Zebrafish Embryos in Vivo.

The aim of this study was to investigate novel chalcones with potent angiogenic activities in vivo. Chalcone-based derivatives were evaluated using a transgenic zebrafish line with fluorescent vessels to real-time monitor the effect on angiogenesis. Results showed that the chalcone analogues did not possess anti-angiogenic effect on zebrafish vasculatures; instead, some of them displayed potent pro-angiogenic effects on the formation of the sub-intestinal vein. Similar pro-angiogenic effects can also be seen on wild type zebrafish embryos. Moreover, the expression of vegfa, the major regulator for angiogenesis, was also upregulated in their treatment. Taken together, we have synthesized and identified a series of novel chalcone-based derivatives as potent in vivo pro-angiogenic compounds. These novel compounds hold potential for therapeutic angiogenesis.

Synthesis of analogues of gingerol and shogaol, the active pungent principles from the rhizomes of Zingiber officinale and evaluation of their anti-platelet aggregation effects.

The present study was aimed at discovering novel biologically active compounds based on the skeletons of gingerol and shogaol, the pungent principles from the rhizomes of Zingiber officinale. Therefore, eight groups of analogues were synthesized and examined for their inhibitory activities of platelet aggregation induced by arachidonic acid, collagen, platelet activating factor, and thrombin. Among the tested compounds, [6]-paradol (5b) exhibited the most significant anti-platelet aggregation activity. It was the most potent candidate, which could be used in further investigation to explore new drug leads.

Toxicity assessments of chalcone and some synthetic chalcone analogues in a zebrafish model.

The aim of this study was to investigate the in vivo toxicities of some novel synthetic chalcones. Chalcone and four chalcone analogues 1a-d were evaluated using zebrafish embryos following antibody staining to visualize their morphological changes and muscle fiber alignment. Results showed that embryos treated with 3'-hydroxychalcone (compound 1b) displayed a high percentage of muscle defects (96.6%), especially myofibril misalignment. Ultrastructural analysis revealed that compound 1b-treated embryos displayed many muscle defect phenotypes, including breakage and collapse of myofibrils, reduced cell numbers, and disorganized thick (myosin) and thin (actin) filaments. Taken together, our results provide in vivo evidence of the myotoxic effects of the synthesized chalcone analogues on developing zebrafish embryos.

Evaluation of the anti-inflammatory effect of chalcone and chalcone analogues in a zebrafish model.

The aim of this study was to investigate novel chalcones with potent anti-inflammatory activities in vivo. Chalcone and two chalcone analogues (compound 5 and 9) were evaluated using a caudal fin-wounded transgenic zebrafish line "Tg(mpx:gfp)" to visualize the effect of neutrophil recruitment dynamically. Results showed that treatment with compound 9 not only affected wound-induced neutrophil recruitment, but also affected Mpx enzymatic activity. Moreover, protein expression levels of pro-inflammatory factors (Mpx, NFκB, and TNFα) were also regulated by compound 9. Taken together, our results provide in vivo evidence of the anti-inflammatory effects of synthesized chalcone analogues on wound-induced inflammation.

The effect of prostacyclin agonists on the differentiation of phorbol ester treated human erythroleukemia cells.

Phorbol-12-myristate-13-acetate (PMA) induces megakaryocytopoeisis in human erythroleukemia (HEL) cells which is characterized by the increase in cell size, increase in nuclear polyploidization and expression of megakaryocyte marker, CD41. However, upon treatment with 100 nM of selective prostacyclin (IP) agonist beraprost inhibits the induced differentiation. Moreover, selective non-prostanoid IP agonist, BMY 45778 prevents PMA induced megakaryocytopoeisis in HEL cells similarly, while prostaglandin E(2) and specific EP(3) agonist sulprostone have no effect. Thus, IP receptor is involved. Furthermore, adenylate cyclase activator forskolin and cAMP analog dibutyryl-cAMP also prevented PMA induced megakaryocytopoeisis in HEL cells. Thus, IP agonists inhibition of PMA induced megakaryocytopoeisis in HEL cells may involve a cAMP dependent pathway.

Inhibition of superoxide anion and elastase release in human neutrophils by 3'-isopropoxychalcone via a cAMP-dependent pathway.

1 Chalcone is abundantly present in the plant kingdom and has various biological activities such as anti-inflammatory and antioxidant. In this study, the semisynthetic chalcone derivative, 3'-isopropoxychalcone (H2O7D), was demonstrated to inhibit the generation of superoxide and the release of elastase, as well as to accelerate resequestration of cytosolic calcium in formyl-L-methionyl-L-leucyl-L-phenylalanine-activated human neutrophils. 2 H2O7D displayed no antioxidant or superoxide-scavenging ability, and it failed to alter the subcellular NADPH oxidase activity. 3 H2O7D induced a substantial increase in cAMP but not cGMP levels. The elevation of cAMP formation by H2O7D was inhibited by adenosine deaminase (ADA). Furthermore, The inhibitory effects of H2O7D were reversed by protein kinase (PK)A inhibitors, as well as ADA and a selective A2a-receptor antagonist. 4 H2O7D inhibited phosphodiesterase (PDE) activities, but it did not alter adenylyl cyclase and soluble guanylyl cyclase activities. These results show that the cAMP-elevating effect of H2O7D results from the inhibition of PDE activity and not from the stimulation of cyclase function. Consistent with this, H2O7D potentiated the PGE(1)-caused inhibitory effects and cAMP formation. 5 In summary, these results indicate that the inhibitory effect of H2O7D is cAMP/PKA dependent, and that it occurs through inhibition of cAMP PDE, which potentiates the autocrine functions of endogenous adenosine. Inhibition of respiratory burst and degranulation in human neutrophils may give this drug the potential to protect against the progression of inflammation.