Effect of a hyaluronan synthase suppressor, 4-methylumbelliferone, on B16F-10 melanoma cell adhesion and locomotion

Hyaluronan (HA) is a ubiquitous, major component of the extracellular matrix. It is involved in cell adhesion and locomotion, and hence in tumor metastasis. We have previously reported that 4-methylumbelliferone (MU) inhibits HA synthesis and may be a useful tool for examining the functions of HA. We here demonstrate that the formation of cell surface HA by melanoma cells and its release into the culture medium are inhibited by MU. Adhesion and locomotion assays revealed that the adhesion and locomotion of melanoma cells were dose-dependently inhibited by MU. Conversely, treatment with exogenous HA enhanced both adhesion and locomotion. Thus, preventing the formation of cell surface HA reduced both the adhesion and locomotion of melanoma cells, suggesting that MU may act as an inhibitor of tumor metastasis.     

Esterified Derivatives of Panaxadiol and Their Inhibitory Effect on HL‐60, THP‐1, and PC‐3 Cell Lines

Panaxadiol is a dammarane‐type ginsenoside having high ginseng content. The 3‐hydroxy group of panaxadiol (PD) was modified by fatty acids and diacids. The modified panax glycol had enhanced anticancer activity. Twelve PD derivatives were evaluated and purified by chemical synthesis, column chromatography, co‐synthesis, and identification. The human leukemia cells THP‐1, HL‐60, and human prostate cancer cell lines PC‐3 were evaluated; PD derivatives were tested and evaluated in vitro by MTT assay. The results showed that the antitumor activities of some derivatives on three tumor cell lines were better than those of PD.     

Effects of coumarin and 7OH-coumarin on bcl-2 and Bax expression in two human lung cancer cell lines in vitro

Coumarin and its derivative 7-hydroxycoumarin (7-OHC) have antitumor and antimetastatic properties. The purpose of this study was to investigate the possible effects of these compounds on expression of the bcl-2 and Bax oncoproteins in two human lung cancer cell lines, A427 and Calu-1. The cells were cultured in vitro for 24 h in RPMI 1640 with 1.5% (v/v) ethanol, 1.0 mM ethanolic coumarin or 1.0 mM ethanolic 7-OHC. Viability was determined in each cell line by an MTT assay. Total protein was extracted from cell lysates and the bcl-2 and Bax oncoproteins were identified. Western blotting showed a decrease in bcl-2 and an increase in Bax in A427 cells cultured with coumarin or 7-OHC. Neither drug changed bcl-2 expression in Calu-1 cells compared to solvent controls, and Bax expression was only slightly increased by coumarin. We conclude that 7-OHC is a more potent inhibitor of cell proliferation than coumarin and has more marked effects on oncoprotein expression. Also, the A427 cell line was more sensitive to the drugs than Calu-1.     

Inhibition of Hyaluronan Synthesis Protects against Central Nervous System (CNS) Autoimmunity and Increases CXCL12 Expression in the Inflamed CNS

Hyaluronan (HA) may have proinflammatory roles in the context of CNS autoimmunity. It accumulates in demyelinated multiple sclerosis (MS) lesions, promotes antigen presentation, and enhances T-cell activation and proliferation. HA facilitates lymphocyte binding to vessels and CNS infiltration at the CNS vascular endothelium. Furthermore, HA signals through Toll-like receptors 2 and 4 to stimulate inflammatory gene expression. We assessed the role of HA in experimental autoimmune encephalomyelitis (EAE), an animal model of MS by administration of 4-methylumbelliferone (4MU), a well established inhibitor of HA synthesis. 4MU decreased hyaluronan synthesis in vitro and in vivo. It was protective in active EAE of C57Bl/6 mice, decreased spinal inflammatory infiltrates and spinal infiltration of Th1 cells, and increased differentiation of regulatory T-cells. In adoptive transfer EAE, feeding of 4MU to donor mice significantly decreased the encephalitogenicity of lymph node cells. The transfer of proteolipid protein (PLP)-stimulated lymph node cells to 4MU-fed mice resulted in a delayed EAE onset and delayed spinal T-cell infiltration. Expression of CXCL12, an anti-inflammatory chemokine, is reduced in MS patients in CSF cells and in spinal cord tissue during EAE. Hyaluronan suppressed production of CXCL12, whereas 4MU increased spinal CXCL12 in naive animals and during neuroinflammation. Neutralization of CXCR4, the most prominent receptor of CXCL12, by administration of AMD3100 diminished the protective impact of 4MU in adoptive transfer EAE. In conclusion, hyaluronan exacerbates CNS autoimmunity, enhances encephalitogenic T-cell responses, and suppresses the protective chemokine CXCL12 in CNS tissue. Inhibition of hyaluronan synthesis with 4MU protects against an animal model of MS and may represent an important therapeutic option in MS and other neuroinflammatory diseases.     

Synthesis,Antioxidant and Antiproliferative Evaluation,Molecular Docking and DFT Studies of Some Novel Coumarin and Fused Coumarin Derivatives

N′-[(4-Chloro-2-oxo-2H-chromen-3-yl)methylene]-2-cyanoacetohydrazide ( 3 ) was synthesized in excellent yield from the condensation of 4-Chloro-2-oxo-2H-chromene-3-carbaldehyde with cyanoacetohydrazide. Compound 3 was utilized as a building block to synthesize novel coumarin and heterocycle-fused coumarin derivatives. The chemical structures of all the new coumarin compounds were identified by spectral analyses. Some of the new coumarins compounds were screened in human cancer cell lines (HEPG-2, MCF-7, HCT-116 and PC-3) to learn about their cytotoxic effects in addition to the study of their DNA damage and antioxidant activity. Three of these compounds exhibited remarkable antioxidant and anti-proliferative activities. Moreover, they have the capability to protect DNA from damage induced by bleomycin. Molecular docking, DFT and molecular electrostatic potential studies were performed on the compounds in vitro.     

Glucosylation of phenolic compounds by Pharbitis nil hairy roots: I. Glucosylation of coumarin and flavone derivatives

Hairy roots of medicinal morning glory (Pharbitis nil) showed potent glucosylation activity against umbelliferone and aesculetin, so the glucosylation activity against several phenolic compounds was tested. Some coumarin derivatives and flavone derivatives having phenolic hydroxyl groups were incubated with the hairy roots. The coumarin derivatives and flavone derivatives almost disappeared from the culture medium in half a day. In the case of the coumarin derivatives, a 7-hydroxyl group was easily glucosylated. A methyl group at C-8 somewhat decreased the glucosylation to a hydroxyl group at C-7 of the coumarin skeleton. The 4-hydroxy coumarin derivatives were changed to acetophenone-type glucosides by incubation with the hairy roots through decarboxylation. Several flavonol derivatives were tested for glucosylation by the hairy roots. 3-Hydroxy flavone, 3.6-dihydroxyflavone and 3,7-dihydroxyflavone were glucosylated to give 3-glucosylated derivatives. Of these, 3,6-dihydroxyflavone was highly glucosylated, but not 3-hydroxyflavone or 3,7-dihydroxyflavone to the same degree. In the case of the flavones, a 3-hydroxy group could be predominantly glucosylated, and hydroxyl groups on the A and B ring of the flavones affected glucosylation by the hairy roots.     

The hyaluronic acid inhibitor 4-methylumbelliferone is an NSMase2 activator—role of Ceramide in MU anti-tumor activity

Increased synthesis of hyaluronic acid (HA) is often associated with increased metastatic potential and invasivity of tumor cells. 4-Methylumbelliferone (MU) is an inhibitor of HA synthesis, and has been studied as a potential anti-tumor drug to inhibit the growth of primary tumors and distant metastasis of tumor cells. Although several studies reported that the anticancer effects of MU are mediated by inhibition of HA signaling, the mechanism still needs to be clarified. In a previous study we demonstrated the regulation of HA synthesis by ceramide, and now show how MU activated neutral sphingomyelinase2 (NSMase2) generates ceramides and mediates MU induced inhibition of HA synthesis, cell migration and invasion, and apoptosis of tumor cells. Using a HA enriched mouse oligodendroglioma cell line G26-24 we found that MU elevated the activity of NSMase2 and increased ceramide levels, which in turn increased phosphatase PP2A activity. Further, the activated PP2A reduced phosphorylation of Akt, decreased activities of HA synthase2 (HAS2) and calpains, and inhibited both the synthesis of HA, and the migration and invasion of G26-24 tumor cells. In addition, MU mediated ceramide stimulated activation of p53 and caspase-3, reduced SIRT1 expression and decreased G26-24 viability. The mechanism of the MU anticancer therefore initially involves NSMase2/ceramide/PP2A/AKT/HAS2/caspase-3/p53/SIRT1 and the calpain signaling pathway, suggesting that ceramides play a key role in the ability of a tumor to become aggressively metastatic and grow.     

Synthesis and cellular characterization of novel isoxazolo- and thiazolohydrazinylidene-chroman-2,4-diones on cancer and non-cancer cell growth and death

Coumarins are extensively studied anticoagulants that exert additional effects such as anticancerogenic and even anti-inflammatory. In order to find new drugs with anticancer activities, we report here the synthesis and the structural analysis of new coumarin derivatives which combine the coumarin core and five member heterocycles in hydrazinylidene-chroman-2,4-diones. The derivatives were prepared by derivatization of the appropriate heterocyclic amines which were used as electrophiles to attack the coumarin ring. The structures were characterized by spectroscopic techniques including IR, NMR, 2D-NMR and MS. These derivatives were further characterized especially in terms of a potential cytotoxic and apoptogenic effect in several cancer cell lines including the breast and prostate cancer cell lines MCF-7, MDA-MB-231, PC-3, LNCaP, and the monocytic leukemia cell line U937. Cell viability was determined after 48 h and 72 h of treatment with the novel compounds by MTT assay and the 50% inhibitory concentrations (EC₅₀ values) were determined. Out of the 8 novel compounds screened for reduced cell viability, 4c, 4d and 4e were found to be the most promising and effective ones having EC₅₀ values that were several fold reduced when compared to the reference substance 4-hydroxycoumarin. However, the effects were cancer cell line dependent. The breast cancer MDA-MB-231 cells, the prostate cancer LNCaP cells, and U937 cells were most sensitive, MCF-7 cells were less sensitive, and PC-3 cells were more resistant. Reduced cell viability was accompanied by increased apoptosis as shown by PARP-1 cleavage and reduced activity of the survival protein kinase Akt.In summary, this study has identified three novel coumarin derivatives that in comparison to 4-hydroxycoumarin have a higher efficiency to reduce cancer cell viability and trigger apoptosis and therefore may represent interesting novel drug candidates.     

DNA‐binding activity and cytotoxic and cell‐cycle arrest properties of some new coumarin derivatives: a multispectral and computational investigation

Coumarins are the most important class of natural compounds found widely in various plants. Many coumarin derivatives with different biological and pharmacological activities have been synthesized. In this study, the antiapoptotic and cytotoxic effects and DNA‐binding properties of some synthetic coumarin derivatives (4b, 4d, 4f, 4 g (DBP‐g), 4 h and 4j) against K562 cell lines were investigated using different techniques. MTT assay indicated that the DBP‐g compound was more active than other derivatives, with a IC₅₀ value of 55 μM, and therefore this compound was chosen for further investigation. Apoptosis induction was assessed using acridine orange/ethidium bromide double‐staining and cell‐cycle analysis. In addition, in vitro DNA‐binding studies were carried out using ultraviolet–visible light absorption and fluorescence spectroscopy, as well as viscosity measurement and molecular modelling studies. In vitro results indicated that DBP‐g interacted with DNA through a groove‐binding mode with a binding constant (K_b) of 1.17 × 10⁴ M^?1. In agreement with other experimental data, molecular docking studies showed that DBP‐g is a minor groove binder. Overall, it can be concluded that DBP‐g could be used as an effective and novel chemotherapeutic agent.     

Design,synthesis and docking study of novel coumarin ligands as potential selective acetylcholinesterase inhibitors

New coumaryl-thiazole derivatives with the acetamide moiety as a linker between the alkyl chains and/or the heterocycle nucleus were synthesized and in vitro tested as acetylcholinesterase (AChE) inhibitors. 2-(diethylamino)-N-(4-(2-oxo-2H-chromen-3-yl)thiazol-2-yl)acetamide (6c, IC₅₀ value of 43?nM) was the best AChE inhibitor with a selectivity index of 4151.16 over BuChE. Kinetic study of AChE inhibition revealed that 6c was a mixed-type inhibitor. Moreover, the result of H4IIE hepatoma cell toxicity assay for 6c showed negligible cell death. Molecular docking studies were also carried out to clarify the inhibition mode of the more active compounds. Best pose of compound 6c is positioned into the active site with the coumarin ring wedged between the residues of the CAS and catalytic triad of AChE. In addition, the coumarin ring is anchored into the gorge of the enzyme by H-bond with Tyr130.     

Water Soluble Coumarin Quaternary Ammonium Chlorides: Synthesis and Biological Evaluation

In the present study, coumarin‐bearing three pyridinium and three tetra‐alkyl ammonium salts were synthesized. The compounds were fully characterized by ¹H‐ and ¹³C‐NMR, LC/MS and IR spectroscopic methods and elemental analyses. The cytotoxic properties of all compounds were tested against human liver cancer (HepG2), human colorectal cancer (Caco‐2) and non‐cancer mouse fibroblast (L‐929) cell lines. Some compounds performed comparable cytotoxicity with standard drug cisplatin. Antibacterial properties of the compounds were tested against Gram‐negative Escherichia coli and Gram‐positive Bacillus subtilis bacteria, but the compounds did not have any antibacterial effect against both bacteria. Enzyme inhibitory properties of all compounds were tested on the activities of human carbonic anhydrase I and II, and xanthine oxidase. All compounds inhibited both enzymes more effectively than standard drugs, acetazolamide and allopurinol, respectively. The biological evaluation results showed that ionic and water soluble coumarin derivatives are promising structures for further investigations especially on enzyme inhibition field.     

Some coumarins and benzoxazinones as potent paraoxonase 1 inhibitors

In this study, we aimed to investigate the effect of some coumarin and benzoxazinone derivatives on the activity of human PON1. Human serum paraoxonase 1 was purified from fresh human serum blood by two-step procedures that are ammonium sulfate precipitation (60–80%) and then hydrophobic interaction chromatography (Sepharose 4B, L-tyrosine and 1-napthylamine). The enzyme was purified 232-fold with a final specific activity of 27.1?U/mg. In vitro effects of some previously synthesized ionic coumarin or benzoxazinone derivatives (1–21) on purified PON1 activity were investigated. Compound 14 (1-(2,3,4,5,6)-pentamethylbenzyl-3-(6,8-dimethyl-2H-chromen-2-one-4-yl))benzimidazolium chloride was found out as the strongest inhibitor (IC₅₀?=?7.84?μM) for PON1 among the compounds. Kinetic investigation and molecular docking study were evaluated for one of the most active compounds (compound 12) and obtained data showed that this compound is competitive inhibitor of PON1 and interact with Leu262 and Ser263 in the active site of PON1. Moreover, coumarin derivatives were found out as the more potent inhibitors for PON1 than benzoxazinone derivatives.     

Synthesis and in vitro antitumour activity of carboplatin analogues containing functional handles compatible for conjugation to drug delivery systems

We describe herein the synthesis of a series of carboplatin derivatives with different functional groups at position 3 of the cyclobutane ring. This pharmacomodulation approach aims at facilitating the vectorisation of these analogues, via their subsequent conjugation to a drug delivery system. Five different derivatives bearing a hydroxy, keto, iodo, azido or amino function at position 3 were synthesised. One of these compounds was coupled to a bifunctional maleimide-containing linker. All compounds were tested in vitro for their cytotoxicity on four different cell lines including two platinum-resistant colorectal cancer cell line (SK-OV-3, HCT116, D3E2, D5B7) using an MTS assay. Overall, the tested compounds were up to six times more potent than carboplatin, especially on D5B7 human colorectal cancer cells. We demonstrated that these modifications led to potent analogues which are compatible with conjugation to a drug delivery system.     

A hyaluronan synthase suppressor, 4-methylumbelliferone, inhibits liver metastasis of melanoma cells

4-Methylumbelliferone (MU) inhibits the cell surface hyaluronan (HA) formation, and that such inhibition results in suppression of adhesion and locomotion of cultured melanoma cells. Here, we examine the effect of MU on melanoma cell metastasis in vivo. MU-treated melanoma cells showed both decreased cell surface HA formation and suppression of liver metastasis after injection into the mice. Oral administration of MU to mice decreased tissue HA content. These HA knock-down mice displayed suppressed liver metastasis. Thus, both cell surface HA of melanoma cells and recipient liver HA can promote liver metastasis, indicating that MU has potential as an anti-metastatic agent.     

Hyaluronan Synthesis Mediates the Fibrotic Response of Keratocytes to Transforming Growth Factor β

TGFβ induces fibrosis in healing corneal wounds, and in vitro corneal keratocytes up-regulate expression of several fibrosis-related genes in response to TGFβ. Hyaluronan (HA) accumulates in healing corneas, and HA synthesis is induced by TGFβ by up-regulation of HA synthase 2. This study tested the hypothesis that HA acts as an extracellular messenger, enhancing specific fibrotic responses of keratocytes to TGFβ. HA synthesis inhibitor 4-methylumbelliferone (4MU) blocked TGFβ induction of HA synthesis in a concentration-dependent manner. 4MU also inhibited TGFβ-induced up-regulation of α-smooth muscle actin, collagen type III, and extra domain A-fibronectin. Chemical analogs of 4MU also inhibited fibrogenic responses in proportion to their inhibition of HA synthesis. 4MU, however, showed no effect on TGFβ induction of luciferase by the 3TP-Lux reporter plasmid. Inhibition of HA using siRNA to HA synthase 2 reduced TGFβ up-regulation of smooth muscle actin, fibronectin, and cell division. Similarly, brief treatment of keratocytes with hyaluronidase reduced TGFβ responses. These results suggest that newly synthesized cell-associated HA acts as an extracellular enhancer of wound healing and fibrosis in keratocytes by augmenting a limited subset of the cellular responses to TGFβ.     

Quinazolin‐4(3H)‐one‐Based Hydroxamic Acids: Design,Synthesis and Evaluation of Histone Deacetylase Inhibitory Effects and Cytotoxicity

The present article describes the synthesis and biological activity of various series of novel hydroxamic acids incorporating quinazolin‐4(3H)‐ones as novel small molecules targeting histone deacetylases. Biological evaluation showed that these hydroxamic acids were potently cytotoxic against three human cancer cell lines (SW620, colon; PC‐3, prostate; NCI?H23, lung). Most compounds displayed superior cytotoxicity than SAHA (suberoylanilide hydroxamic acid, Vorinostat) in term of cytotoxicity. Especially, N‐hydroxy‐7‐(7‐methyl‐4‐oxoquinazolin‐3(4H)‐yl)heptanamide ( 5b ) and N‐hydroxy‐7‐(6‐methyl‐4‐oxoquinazolin‐3(4H)‐yl)heptanamide ( 5c ) (IC₅₀ values, 0.10–0.16 μm ) were found to be approximately 30‐fold more cytotoxic than SAHA (IC₅₀ values of 3.29–3.67 μm ). N‐Hydroxy‐7‐(4‐oxoquinazolin‐3(4H)‐yl)heptanamide ( 5a ; IC₅₀ values of 0.21–0.38 μm ) was approximately 10‐ to 15‐fold more potent than SAHA in cytotoxicity assay. These compounds also showed comparable HDAC inhibition potency with IC₅₀ values in sub‐micromolar ranges. Molecular docking experiments indicated that most compounds, as represented by 5b and 5c , strictly bound to HDAC2 at the active binding site with binding affinities much higher than that of SAHA.     

Identification of aromatic dihydroxy acids in biological fluids

3,5-Dihydroxyphenylpropionic acid, 3,5-dihydroxycinnamic acid and 2,3-dihydroxycinnamic acid were detected for the first time to be components of human urine. In the course of this investigation all constitutional isomers of dihydroxy-benzoic, -phenylpropionic, -phenylacetic and -cinnamic acid were synthesized. Mass spectra and retention indices of methyl and trimethylsilyl (TMS) derivatives were determined. In contrast to many other substituted aromatic compounds the mass spectra of methyl and TMS derivatives of dihydroxy aromatic acids often allow a firm distinction to be made between constitutional isomers: TMS derivatives of aromatic acids containing two hydroxy groups located in the ortho position to each other can be recognized by ions resulting from a primary cleavage reaction mainly in the side chain or ester group, followed by loss of tetramethylsilane. In methyl derivatives of 1,2,3-trisubstituted isomers, methoxy groups are lost much more easily from the ions corresponding to the benzylic cleavage than in other isomers. Methyl derivatives of dihydroxycinnamic acids containing at least one methoxy group in the ortho position to the side chain are characterized by a fragmentation reaction, corresponding to the loss of dimethyl ether. TMS and methyl derivatives of 3,5-dihydroxy aromatic acids show unique structure-specific fragmentation reactions.     

Synthesis and evaluation of novel fluorogenic substrates for the detection of bacterial beta-galactosidase

AIMS: A widely used coumarin derivative is 7-hydroxy-4-methylcoumarin-beta-D-galactoside (4-methylumbelliferone-beta-D-galactoside; 4-MU-GAL). This galactoside is utilized as a substrate for the detection of the beta-galactosidase activity of coliform bacteria in water analysis. The intense fluorescence of coumarin-based molecules has enabled them to be incorporated into enzyme-based tests for the quantitative assay of indicator bacteria. The aim of this present study was to evaluate the potential of other coumarin derivatives, by synthesis of a selection of core coumarin molecules. METHODS AND RESULTS: Several coumarin derivatives were found to be more promising than 4-MU, with ethyl-7-hydroxycoumarin-3-carboxylate (EHC) giving a combination of greater fluorescence over a broad pH range and reduced growth inhibition with 12 representative coliform strains. On conversion to a beta-galactoside derivative, EHC-GAL generated a more rapid fluorescence than any other tested substrate. CONCLUSIONS: When tested in a broth assay format, based on most probable number (MPN), low numbers of coliforms were detected with EHC-GAL around 1 h earlier than with 4-MU-GAL. SIGNIFICANCE AND IMPACT OF THE STUDY: The present study suggests that EHC-GAL should be evaluated as a substrate for the detection of coliforms in water analysis, due to a combination of the following favourable features: (i) reduced toxicity; (ii) increased fluorescence; (iii) pH stability of fluorescence; and (iv) rapid detection.     

Hyaluronan expressed by the hematopoietic microenvironment is required for bone marrow hematopoiesis

The contribution of hyaluronan (HA) to the regulatory network of the hematopoietic microenvironment was studied using knock-out mice of three hyaluronan synthase genes (Has1, Has2, and Has3). The number of hematopoietic progenitors was decreased in bone marrow and increased in extramedullary sites of Prx1-Cre;Has2(flox/flox);Has1(-/-);Has3(-/-) triple knock-out (tKO) mice as compared with wild type (WT) and Has1(-/-);Has3(-/-) double knock-out (dKO) mice. In line with this observation, decreased hematopoietic activity was observed in long term bone marrow cultures (LTBMC) from tKO mice, whereas the formation of the adherent layer and generation of hematopoietic cells in WT and dKO cultures was not different. 4-Methylumbelliferone (4MU) was used to pharmacologically inhibit the production of HA in LTBMC. Treatment with 4MU inhibited HA synthesis, decreased expression of HAS2 and HAS3, and eliminated hematopoiesis in LTBMC, and this effect was alleviated by the addition of exogenous HA. Exogenous HA also augmented the cell motility in LTBMC, which correlated with the HA-stimulated production of chemokines and growth factors. Conditioned media from HA-induced LTBMC enhanced the chemotaxis of hematopoietic stem/progenitor cells (HSPC) in response to SDF-1. Exposure of endothelial cells to 4MU decreased their ability to support HSPC rolling and adhesion. In addition, migration of transplanted HSPC into the marrow of 4MU-pretreated mice was lower than in untreated mice. Collectively, the results suggest that HA depletion reduces the ability of the microenvironment to support HSPC, and confirm a role for HA as a necessary regulatory element in the structure of the hematopoietic microenvironment.