EC3, a novel heterodimeric disintegrin from Echis carinatus venom, inhibits alpha4 and alpha5 integrins in an RGD-independent manner

EC3, a heterodimeric disintegrin (Mr = 14,762) isolated from Echis carinatus venom is a potent antagonist of alpha4 integrins. Two subunits called EC3A and EC3B were isolated from reduced and alkylated EC3 by reverse-phase high performance liquid chromatography. Each subunit contained 67 residues, including 10 cysteines, and displayed a high degree of homology to each other and to other disintegrins. EC3 inhibited adhesion of cells expressing alpha4beta1 and alpha4beta7 integrins to natural ligands vascular cell adhesion molecule 1 (VCAM-1) and mucosal addressin cell adhesion molecule 1 (MadCAM-1) with IC50 = 6-30 nM, adhesion of K562 cells (alpha5beta1) to fibronectin with IC50 = 150 nM, and adhesion of alphaIIbbeta3 Chinese hamster ovary cells to fibrinogen with IC50 = 500 nM; it did not inhibit adhesion of alphavbeta3 Chinese hamster ovary cells to vitronectin. Ethylpyridylethylated EC3B inhibited adhesion of Jurkat cells to immobilized VCAM-1 (IC50 = 6 microM), whereas EC3A was inactive in this system. The MLDG motif appeared to be essential for activity of EC3B. Linear MLDG peptide inhibited the adhesion of Jurkat to VCAM-1 in a dose-dependent manner (IC50 = 4 mM), whereas RGDS peptide was not active at the same concentration. MLDG partially inhibited adhesion of K562 cells to fibronectin (5-10 mM) in contrast to RGDS peptide (IC50 = 3 mM), inhibiting completely at 10 mM.     

Inhibitory activity of linoleic acid isolated from proso and Japanese millet toward histone deacetylase

Linoleic acid was isolated from both the methanol extracts of proso and Japanese millet as a histone deacetylase inhibitor. It showed uncompetitive inhibitory activity toward histone deacetylase (IC(50)=0.51 mM) and potent cytotoxicity toward human leukemia K562 (IC(50)=68 microM) and prostate cancer LNCaP cells (IC(50)=193 microM). Millet containing linoleic acid might have anti-tumor activity.     

A sugar ester and an iridoid glycoside from Scrophularia ningpoensis

From cytotoxic extracts of the roots of Scrophularia ningpoensis Hemsl. (Scrophulariaceae) a new sugar ester, ningposide D (3-O-acetyl-2-O-p-methoxycinnamoyl-alpha(beta)-L-rhamnopyranose) (1) and a new iridoid glycoside, scrophuloside B4 (6-O-(2'-O-acetyl-3'-O-cinnamoyl-4'-O-p-methoxycinnamoyl-alpha-L-rhamnopyranosyl) catalpol) (2) along with known compounds: oleanonic acid (3), ursolonic acid (4), cinnamic acid (5), 3-hydroxy-4-methoxy benzoic acid (6), 5-(hydroxymethyl)-2-furfural (7) and beta-sitosterol (8) were isolated. The structures of the new compounds were elucidated by spectral data (1, 2D NMR, EI, HRESI-MS and MS/MS). Oleanonic acid (3) and ursolonic acid (4) were found to be cytotoxic against a series of human cancer cell lines with IC50=4.6, 15.5 microM on MCF7; 4.2, 14.5 microM on K562; 14.8, 44.4 microM on Bowes; 24.9, 43.6 microM on T24S; 61.3, 151.5 microM on A549, respectively. Beta-sitosterol (8) inhibited Bowes cells growth at IC50=36.5 microM. Scrophuloside B4 (2) showed activity on K562 and Bowes cells at IC50=44.6, 90.2 microM, respectively.     

The total synthesis of an aurone isolated from Uvaria hamiltonii: aurones and flavones as anticancer agents

The naturally occurring aurone 1, isolated from Uvaria hamiltonii, and a series of aurones analogues based structurally on known tubulin binding agents were prepared and evaluated for anticancer activity. Aurone 20 was the most active (IC(50) K562 50 nM) and caused significant G2/M cell-cycle arrest.     

Design, synthesis and biological evaluation of novel acrylamide analogues as inhibitors of BCR-ABL kinase

A series of acrylamide analogues were designed and synthesized from Imatinib and Nilotinib as novel BCR-ABL inhibitors by application of the principle of nonclassical electronic isostere. All new compounds were evaluated for their inhibitory effects on the activity of BCR-ABL kinase and the proliferation of K562 leukemia cancer cells in vitro. The acrylamide analogues in which the substituent in C ring was trifluoromethyl group were identified as highly potent BCR-ABL kinase inhibitors. Compound 13f exhibited an IC(50) value as low as 20.6nM in ABL kinase inhibition and an IC(50) value of 32.3nM for antiproliferative activity, about 10.5-fold and 12-fold lower than those of Imatinib respectively. These results suggest that compound 13f is a promising candidate as a novel BCR-ABL kinase inhibitor for further development.     

BaG,a new dimeric metalloproteinase/disintegrin from the Bothrops alternatus snake venom that interacts with alpha5beta1 integrin

The alpha(5)beta(1) integrin is one of the major fibronectin receptors which plays an essential role in the adhesion of normal and tumor cells to extracellular matrix. Here, we describe the isolation and characterization of a novel dimeric metalloproteinase/disintegrin, which is an inhibitor of fibronectin binding to the alpha(5)beta(1) integrin. This protein (BaG) was isolated from the venom of the South American snake Bothrops alternatus by gelatin-Sepharose affinity and anion exchange chromatography. The molecular mass of BaG was approximately 130 kDa under non-reducing conditions and 55 kDa under reducing conditions by SDS-PAGE. BaG shows proteolytic activity on casein that was inhibited by EDTA. 1,10-phenanthroline-treated BaG (BaG-I) inhibits ADP-induced platelet aggregation with an IC(50) of 190 nM. BaG-I inhibits fibronectin-mediated K562 cell adhesion with an IC(50) of 3.75 microM. K562 cells bind to BaG-I probably through interaction with alpha(5)beta(1) integrin, since anti-alpha(5)beta(1) antibodies inhibited K562 cell adhesion to BaG-I. In addition, BaG-I induces the detachment of K562 cells that were bound to fibronectin. In summary, we have purified a novel, dimeric snake venom metalloproteinase/disintegrin that binds to the alpha(5)beta(1) integrin.     

A phenolic compound, 5-caffeoylquinic acid (chlorogenic acid), is a new type and strong matrix metalloproteinase-9 inhibitor: isolation and identification from methanol extract of Euonymus alatus

A phenolic compound responsible for anti-MMP-9, which is known to be involved in tumor cell invasion and metastasis, has been isolated from methanol extracts prepared from stem barks of Euonymus alatus by assay-guided fractionation. The compound has been identified as 5-caffeoylquinic acid (chlorogenic acid; CHA) by NMR and FAB-MS. CHA showed a strong inhibitory effect of matrix metalloproteinase (MMP)-9 activity in a concentration-dependent manner on zymography. The purified CHA inhibited MMP-9 activity with the IC50 of 30-50 nM. Furthermore, the cytotoxic survival curve showed that CHA does not have cytotoxic effects on cellular proliferation, when Hep3B cells were treated with various concentrations of CHA and cell viability was measured using the XTT assay. The present data suggest a clue for possible mechanisms of cancer chemoprevention by CHA and other naturally occurring phenolic compounds. The results also imply that useful cancer chemopreventive agents can be further identified by combinations of in vitro (as a first screen) and in vivo studies.     

Induction of apoptosis against cancer cell lines by four ascomycetes (endophytes) from Malaysian rainforest

Endophytic fungi have been shown to be a promising source of biologically active natural products. In the present study, extracts of four endophytic fungi isolated from plants of the National Park, Pahang were evaluated for their cytotoxic activity and the nature of their active compounds determined. Those extracts exhibiting activity with IC(50) values less than 17 μg/ml against HCT116, MCF-7 and K562 cell lines were shown to induce apoptosis in these cell lines. Molecular analysis, based on sequences of the rDNA internal transcribed spacers ITS1 and ITS4, revealed all four endophytic fungi to be ascomycetes: three sordariomycetes and a dothideomycete. Six known compounds, cytochalasin J, dechlorogriseofulvin, demethylharzianic-acid, griseofulvin, harzianic acid and 2-hexylidene-3-methyl-succinic acid were identified from a rapid dereplication technique for fungal metabolites using an in-house UV library. The results from the present study suggest the potential of endophytic fungi as cytotoxic agents, and there is an indication that the isolates contain bioactive compounds that mainly kill cancer cells by apoptosis.     

A diterpene quinone from the bark of Cryptomeria japonica

A diterpene, cryptoquinone, was isolated from the bark of Cryptomeria japonica, the structure, 7,11,14-trioxoabieta-8,12-diene, was established by spectral analyses and X-ray crystallography. This diterpene quinone showed moderate antifungal activities against Pyricularia orizae and Alternaria alternata, and cytotoxic activity against mouse lymphoid neoplasm (P388) cells with an IC(50) of 0.26 microg/ml.     

Cytotoxicity of a new IMP dehydrogenase inhibitor, benzamide riboside, to human myelogenous leukemia K562 cells.

COMPARE computer program suggested that benzamide riboside, BR, 3-(1-deoxy-beta-D-ribofuranosyl)benzamide, should have a similar mechanism of action as that of tiazofurin, an inhibitor of IMP dehydrogenase (IMPDH). This hypothesis was tested in K562 cells in culture. BR was cytotoxic to K562 cells with an IC50 of 2 microM. Incubation of K562 cells with BR resulted in a significant decrease in GMP and GTP levels with a concurrent increase in IMP pools, and with a significant inhibition of IMPDH activity. However, 290-fold higher BR concentration was needed to demonstrate in vitro inhibition of IMPDH activity, suggesting that the agent may require metabolism to exert its action. These results provide evidence that BR is a new inhibitor of IMPDH. This investigation should be helpful to design new analogues having activity against IMPDH.     

New quinoline/chalcone hybrids as anti-cancer agents: Design,synthesis, and evaluations of cytotoxicity and PI3K inhibitory activity

A series of quinoline-chalcone hybrids was designed as potential anti-cancer agents, synthesized and evaluated. Different cytotoxic assays revealed that compounds experienced promising activity. Compounds 9i and 9j were the most potent against all the cell lines tested with IC₅₀ = 1.91–5.29 µM against A549 and K-562 cells. Mechanistically, 9i and 9j induced G₂/M cell cycle arrest and apoptosis in both A549 and K562 cells. Moreover, all PI3K isoforms were inhibited non selectively with IC₅₀s of 52–473 nM when tested against the two mentioned compounds with 9i being most potent against PI3K-γ (IC₅₀ = 52 nM). Docking of 9i and 9j showed a possible formation of H-bonding with essential valine residues in the active site of PI3K-γ isoform. Meanwhile, Western blotting analysis revealed that 9i and 9j inhibited the phosphorylation of PI3K, Akt, mTOR, as well as GSK-3β in both A549 and K562 cells, suggesting the correlation of blocking PI3K/Akt/mTOR pathway with the above antitumor activities. Together, our findings support the antitumor potential of quinoline-chalcone derivatives for NSCLC and CML by inhibiting the PI3K/Akt/mTOR pathway.     

Assay for determination of daunorubicin in cancer cells with multidrug resistance phenotype

A sensitive assay for direct determination of intracellular level of daunorubicin (DRN) in resistant leukemia cells with overexpressed P-glycoprotein has been developed. This assay is based on a rapid separation of cells from media and fast cut-off of DRN transportation by centrifugation of cells through a layer of silicone oil. Cell pellets were extracted using 1% (v/v) formic acid in 50% (v/v) ethanol in water. The cell extracts were subsequently analysed by liquid chromatography (HPLC) coupled a low-energy collision tandem mass spectrometer equipped with an electrospray ionization source (ESI-CID-MS/MS) operated in the multiple-reaction monitoring (MRM) mode. Calibration curve was linear from 0.4 to 250nM with correlation coefficient (r2) better than 0.998. The limit of quantitation (LOQ) was 0.4 nM. The assay has been successfully applied to a determination of intracellular content of daunorubicin in sensitive K562 and resistant K562/Dox and K562/HHT300 cells.     

Synthesis, in vitro anticancer evaluation, and interference with cell cycle progression of N-phosphoamino acid esters of zidovudine and stavudine

A series of N-diisopropylphosphoryl (DIPP) L-amino acid ester prodrugs of zidovudine (AZT) (3a-3e) and stavudine (d4T) (4a-4e) has been prepared. The activity of these compounds against MCF-7 cells (human pleural effusion breast adenocarcinoma cell line) and K562 cells (human chronic myeloid leukemia (CML) cell line) was evaluated. In difference from that of AZT amino acid phosphoramidates, the alophatic amino acid esters of AZT were found to be more cytotoxic than the aromatic analogues toward MCF-7 cell. Two DIPP-L-amino acid esters of d4T 4b (CC50 = 83 microM) and 4c (CC50 = 182 microM) were found to be more cytotoxic than the parent drug toward K562 cells. MCF-7 and K562 cell cycle disturbance was investigated showing detectable blockade in the S phase when exposed to biologically active AZT, 3a, 3b, 3c, 4b and 4c, indicating that they inhibit cell growth by blocking cell cycle progression. Together with previous reports, present findings suggest that anti-breast cancer activity of AZT may be due to hamper DNA synthesis.     

Antibacterial and cytotoxic activities of extracts from (Tunisian)Rhamnus alaternus (Rhamnaceae)

The petroleum ether, chloroformic, ethyl acetate, methanolic, Total Oligomers Flavonoids (TOF) enriched extracts, water extract as well as its fractions A₁, A₂, A₃ obtained from aerial parts ofRhamnus alaternus, a Tunisian-Mediterranean medicinal species, were investigated for the contents of phenolic compounds, cytotoxic activity against the K562 human chronic myelogenous leukaemia cell line and L1210 leukaemia murine cells and for antibacterial activity against Gram positive and Gram negative bacterial reference strains. A pronounced cytotoxic effect on both the cell lines was shown in the TOF, ethyl acetate, methanolic, aqueous extracts and A₂ fraction, with respectively IC₅₀ values 75, 232, 298, 606 and 571 μg/ml on K562 cells and 198, 176, 767, 560 and 614 μg/ml on L1210 cell line. Significant activity against bacterial reference strains:Staphylococcus aureus, Enterococcus faecalis, Escherichia coli, Salmonella enteritidis andSalmonella typhimurium was shown with ethyl acetate, TOF extracts and A2 fraction. The antimicrobial and cytotoxic activities showed byR. alatemus depended on the chemical composition of the tested extracts.     

The in vitro effect of new muramyl peptide derivatives on cytotoxic activity of NK (Natural Killer) cells from hamsters bearing AB Bomirski Melanoma

The modulation of NK activity by muramyl dipeptides derivatives against Ab (amelanotic) Bomirski melanoma and human erythroleukemia K562 cells was studied in vitro. The stimulatory effect was observed for 3 of 7 muramyl dipeptides: MDP(L-Ala)C921, MDPC857 and L18-MDP(Ala) in relation to cytotoxic activity of NK cells obtained from peripheral blood and spleen of healthy and Ab Bomirski melanoma bearing hamsters. An increased of cytotoxic activity NK cells isolated from animals before and during the transplantable phase of the tumor against K562 was found. A similar stimulation was received for NK cells obtained from animals against their own melanoma cells. The most significant influence of examined MDP derivatives on the cytotoxic activity of NK cells were obtained from animals between 10 to 12 days of tumor growth. The extent of the modulation of cytotoxic activity of NK cells was dependent on its initial value both in healthy control and Ab Bomirski melanoma bearing hamsters. If natural cytotoxic activity was high the stimulatory effect of the examined MDP derivatives was only slightly expressed.     

Antimitotic and cell growth inhibitory properties of combretastatin A-4-like ethers

A series of diarylamines, diaryl and arylbenzyl ethers based on combretastatin A-4 was prepared and evaluated for anticancer activity. 2-Methoxy-5-(3',4',5'-trimethoxyphenoxymethyl)phenol was the most active (IC50, K562 20 nM) and caused significant G2/M cell cycle arrest.     

The bisabolane sesquiterpenoid endoperoxide, 3,6-epidioxy-1,10-bisaboladiene, isolated from Cacalia delphiniifolia inhibits the growth of human cancer cells and induces apoptosis

In the course of our screening for a new anti-tumor substance, the bisabolane sesquiterpenoid endoperoxide, 3,6-epidioxy-1,10-bisaboladiene (EDBD), was isolated from the edible wild-plant, Cacalia delphiniifolia. EDBD showed cytotoxicity toward human chronic myelogenous leukemia K562 and human prostate carcinoma LNCaP cell lines with IC50 values of 9.1 microM and 23.4 microM, respectively. DNA fragmentation and condensation of chromatin, the hallmarks of apoptosis, appeared in K562 cells after an 18-h treatment with EDBD. alpha-Curcumene, a bisabolane sesquiterpene that lacks the endoperoxide moiety of EDBD, also showed cytotoxicity toward both K562 and LNCaP cell lines at over a 10-times higher dose than that of EDBD. The results indicate the importance of the endoperoxide structure within EDBD to its anti-tumor activity in vitro.     

Identification of high levels of type 1 and type 2A protein phosphatases in higher plants.

Extracts of Brassica napus (oilseed rape) seeds contain type 1 and type 2A protein phosphatases whose properties are indistinguishable from the corresponding enzymes in mammalian tissues. The type 1 activity dephosphorylated the beta-subunit of phosphorylase kinase selectively and was inhibited by the same concentrations of okadaic acid [IC50 (concentration causing 50% inhibition) approximately 10 nM], mammalian inhibitor 1 (IC50 = 0.6 nM) and mammalian inhibitor 2 (IC50 = 2.0 nM) as the rabbit muscle type 1 phosphatase. The plant type 2A activity dephosphorylated the alpha-subunit of phosphorylase kinase preferentially, was exquisitely sensitive to okadaic acid (IC50 approximately 0.1 nM), and was unaffected by inhibitors 1 and 2. As in mammalian tissues, a substantial proportion of plant type 1 phosphatase activity (40%) was particulate, whereas plant type 2A phosphatase was cytosolic. The specific activities of the plant type 1 and type 2A phosphatases were as high as in mammalian tissue extracts, but no type 2B or type 2C phosphatase activity was detected. The results demonstrate that the improved procedure for identifying and quantifying protein phosphatases in animal cells is applicable to higher plants, and suggests that okadaic acid may provide a new method for identifying plant enzymes that are regulated by reversible phosphorylation.