Effect of an NGR Peptide on the Efficacy of the Doxorubicin Phospholipid Delivery System.

This study is a continuation of an investigation into the effect of a targeted component, a peptide with an NGR, on the properties of the previously developed doxorubicin phospholipid delivery system. The NGR peptide has an affinity for aminopeptidase N (known as the CD13 marker on the membrane surface of tumor cells) and has been extensively used to target drug delivery systems. This article presents the results of a study investigating the physical properties of the phospholipid composition with and without the peptide chain: particle size, zeta potential, stability in fluids, and dependence of doxorubicin release from nanoparticles at different pH levels (5.0, 6.5, 7.4). The cytotoxic effect of the compositions has also been shown to depend on the dose of the drug used for incubation, the presence of the targeted component in the composition, and the time of incubation time of the substances. There was a significant difference in the cytotoxic effect on HT-1080 (CD13-positive) and MCF-7 (CD13-negative) cells. Cell death pathway analysis has shown that death occurred mainly by apoptosis. We also present data on the effect of doxorubicin embedded in phospholipid nanoparticles with the targeted peptide on DNA assessed by differential pulse voltammetry, the mechanism of action being electrostatic interactions. The interactions of native dsDNA with doxorubicin encapsulated in phospholipid nanoparticles with the targeted peptide were studied electrochemically by differential pulse voltammetry. Here, we have highlighted that the targeted peptide in the doxorubicin composition moved specific interaction of the drug with dsDNA from intercalative mode to electrostatic interactions.

Oxazolinyl derivatives of androst-16-ene as inhibitors of CYP17A1 activity and prostate carcinoma cells proliferation: Effects of substituents in oxazolinyl moiety.

Steroid derivatives modified with nitrogen containing heterocycles are known to inhibit activity of steroidogenic enzymes, decrease proliferation of cancer cells and attract attention as promising anticancer agents. Specifically, 2'-(3ß-hydroxyandrosta-5,16-dien-17-yl)-4',5'-dihydro-1',3'-oxazole 1a potently inhibited proliferation of prostate carcinoma cells. In this study we synthesized and investigated five new derivatives of 3ß-hydroxyandrosta-5,16-diene comprising 4'-methyl or 4'-phenyl substituted oxazolinyl cycle 1 (b-f). Docking of compounds 1 (a-f) to CYP17A1 active site revealed that the presence of substitutents at C4' atom in oxazoline cycle, as well as C4' atom configuration, significantly affect docking poses of compounds in the complexes with enzyme. Testing of compounds 1 (a-f) as CYP17A1 inhibitors revealed that the only compound 1a, comprising unsubstituted oxazolinyl moiety, demonstrated strong inhibitory activity, while other compounds 1 (b-f) were slightly active or non active. Compounds 1 (a-f) efficiently decreased growth and proliferation of prostate carcinoma LNCaP and PC-3 cells at 96 h incubation; the effect of compound 1a was the most powerful. Compound 1a efficiently stimulated apoptosis and caused PC-3 cells death, that was demonstrated by a direct comparison of pro-apoptotic effects of compound 1a and abiraterone.

Chlorin e6 Phospholipid Delivery System Featuring APN/CD13 Targeting Peptides: Cell Death Pathways, Cell Localization, In Vivo Biodistribution.

We have previously designed a phospholipid delivery system for chlorin e6 to increase the efficacy of photodynamic therapy involving a second-generation photosensitizer. Further research into the matter led to double modification of the obtained nanoparticles with ligands exhibiting targeting and cell-penetrating effects: an NGR-containing peptide and heptaarginine (R7), respectively. This study investigated the cell death pathway on HT-1080 tumor cells after treatment with the proposed compositions: the chlorin e6 phospholipid composition and the two-peptide chlorin e6 phospholipid composition. It was demonstrated that most of the cells died by apoptosis. Colocalization analysis of chlorin e6 in the phospholipid composition with two peptides showed mitochondria are one of the targets of the photosensitizer. An HT-1080 tumor-bearing mouse model was used to evaluate the biodistribution of the drug in tumor, liver, and kidney tissues after administration of the study compositions in comparison with free chlorin e6. The photosensitizer mostly accumulated in the tumor tissue of mice administered the phospholipid compositions, and accumulation was increased 2-fold with the peptide-containing composition and approximately 1.5-fold with the unenhanced composition, as compared with free chlorin e6. The enhancement of the chlorin e6 phospholipid composition with targeting and cell-penetrating peptides was found to be effective both in vitro and in vivo.

Chondroitin Sulfate and Fucosylated Chondroitin Sulfate as Stimulators of Hematopoiesis in Cyclophosphamide-Induced Mice.

The immunosuppression and inhibition of hematopoiesis are considered to be reasons for the development of complications after intensive chemotherapy and allogeneic hematopoietic stem cell transplantation. Chondroitin sulfate (CS), isolated from the fish Salmo salar, and fucosylated chondroitin sulfate (FCS), isolated from the sea cucumber Apostichopus japonicus, were studied for their roles as stimulators of hematopoiesis in a model of cyclophosphamide-induced immunosuppression in mice. The recombinant protein r G-CSF was applied as a reference. The studied polysaccharides were shown to stimulate the release of white and red blood cells, as well as platelets from bone marrow in immunosuppressed mice, while r G-CSF was only responsible for the significant increase in the level of leucocytes. The analysis of different populations of leucocytes in blood indicated that r G-CSF mainly stimulated the production of neutrophils, whereas in the cases of the studied saccharides, increases in the levels of monocytes, lymphocytes and neutrophils were observed. The normalization of the level of the pro-inflammatory cytokine IL-6 in the serum and the recovery of cell populations in the spleen were observed in immunosuppressed mice following treatment with the polysaccharides. An increase in the proliferative activity of hematopoietic cells CD34(+)CD45(+) was observed following ex vivo polysaccharide exposure. Further study on related oligosaccharides regarding their potential as promising drugs in the complex prophylaxis and therapy of hematopoiesis inhibition after intensive chemotherapy and allogeneic hematopoietic stem cell transplantation seems to be warranted.

Implication of integrin α2ß1 in senescence of SK-Mel-147 human melanoma cells.

This investigation addressed the impact of integrin-initiated signaling pathways on senescence of tumor cells. In a model of human SK-Mel-147 melanoma cells, the silencing of integrin α2ß1 strongly reduced cell proliferation and enhanced the percentage of SA-ß-Gal-positive cells, a phenotypic feature of cellular senescence. These changes were accompanied by a significant increase in the activity of Akt and mTOR protein kinases and also in the expression of p53 and p21 oncosuppressors. Pharmacological inhibition of Akt and mTORC1 and genetic inhibition of p53 and p21 reduced the senescence of α2ß1-depleted SK-Mel-147 cells to the level of control cells. Based on our earlier data on the non-canonical functions of Akt isomers in the invasion and anoikis of SK-Mel-147 cells, we investigated the role of Akt isomers in senescence induced by α2ß1 suppression. The inhibition of Akt1 strongly reduced the percentage of SA-ß-Gal-positive cells in the α2ß1-depleted cell population, while the inhibition of Akt2 did not have a noticeable effect. Our data demonstrated for the first time that α2ß1 is involved in the protection of tumor cells against senescence and that senescence, which is induced by the downregulation of α2ß, is based on a signaling mechanism in which Akt1 performs a non-canonical function.

High-density lipoprotein remodeling by phospholipid nanoparticles improves cholesterol efflux capacity and protects from atherosclerosis.

The efficiency of cholesterol efflux from cells promoted by high-density lipoproteins (HDLs) depends on HDL concentration and functional properties. The term "dysfunctional HDL" describes HDLs with impaired protective properties. Cholesterol efflux capacity (CEC) of HDL is reduced in patients with atherosclerosis, but the exact mechanisms underlying this impairment are not well characterized. Enriching HDLs with phospholipids (PLs) improves CEC. Herein, we assessed the potential of PL nanoparticles in improving HDL functionality. We lipidated HDL subfractions by incubating with PL nanoparticles containing soybean polyunsaturated phosphatidylcholine. Incubating blood plasma with PL nanoparticles resulted in the dose-dependent lipidation of all HDL subfractions. Changes in apolipoprotein A1 (apoA-1) and PL concentrations were the most prominent in the HDL2 fraction. Concentrations of PL in the HDL3 fraction and the fraction with a density > 1.21 g/mL increased by 30-50%, whereas apoA-1 levels decreased. We hypothesized that PL nanoparticles may cause HDL remodeling that can improve their functions. The CECs of lipidated HDLs were analyzed by incubating apolipoprotein B (apoB)-depleted plasma with 3H-cholesterol-labeled THP-1 macrophages. The findings revealed a two-fold increase in cholesterol efflux compared with native apoB-depleted plasma. Moreover, intravenous administration of PL nanoparticles restored lipid profiles and effectively protected blood vessels from atherosclerosis progression in cholesterol-fed rabbits compared with that of fenofibrate and atorvastatin. PL nanoparticles also protected against atherosclerosis and decreased the atherogenic index. Altogether, these results indicate that PL nanoparticles can be used to correct the lipid composition and CEC of HDLs. DATA AVAILABILITY: Additional data can be provided upon reasonable request from the date of publication of this article within 5 years. The request should be sent to the author-correspondent at the address cd95@mail.ru.

Chlorin e6 embedded in phospholipid nanoparticles equipped with specific peptides: Interaction with tumor cells with different aminopeptidase N expression.

A promising direction in Biopharmaceuticals is the development of specific peptide-based systems to improve drug delivery. This approach may increase tumor specificity and drug penetration into the target cell. Similar systems have been designed for several antitumor drugs. However, for photodynamic therapy drugs, such studies are not yet enough. Previously, we have developed a method of inclusion of chlorin e6 (Ce6), a photosensitizer used in photodynamic therapy, in phospholipid nanoparticles with a diameter of up to 30 nm, and reported an increase in its effectiveness in the experiments in vivo. In this work, we propose to modify a previously developed delivery system for Ce6 by the addition of cell-penetrating (R7) and/or targeting NGR peptides. The interaction of the compositions developed with HepG2 and MCF-7 tumor cells is shown. The expression of CD13 protein with affinity to NGR on the surface of these cells has been studied using flow cytometry. The expression of this protein on the HepG2 cells and its absence on MCF-7 was demonstrated. After incubation of tumor cells with the resulting Ce6 compositions, we evaluated the cellular accumulation, photoinduced, and dark cytotoxicity of the drugs. After irradiation, the highest level of cytotoxicity was observed when R7 peptide was added to the system, either alone or in combination with NGR. In addition to R7, the NGR-motif peptide increased the internalization of Ce6 in HepG2 cells without affecting its photodynamic activity. In this work we also discuss possible mechanisms of action of the cell-penetrating peptide when attached to phospholipid nanoparticles.

Implication of integrins α3ß1 and α5ß1 in invasion and anoikis of SK-Mel-147 human melanoma cells: non-canonical functions of protein kinase Akt.

Downregulation of integrins α3ß1 and α5ß1 strongly decreased cell colony formation and in vitro invasion and markedly enhanced anoikis in SK-Mel-147 human melanoma cells. These modifications were accompanied by a marked increase in the levels of active Akt protein kinase, which indicated it played a non-canonical function in the melanoma cells. Pharmacological inhibition of Akt1, an Akt isozyme, in cells depleted of α3ß1 or α5ß1 restored their invasive activity, while inhibition of the Akt 2 isoform did not cause a visible effect. Similar to our previous results with the α2ß1 integrin, this finding suggested that in signaling pathways initiated by α3ß1 and α5ß1, the Akt1 isoform performs a non-canonical function in regulating invasive phenotype of melanoma cells. In contrast, when the effects of Akt inhibitors on anoikis of the melanoma cells were compared, the Akt2 isoform demonstrated a non-canonical activity in which Akt2 suppression led to a significant attenuation of apoptosis in cells with downregulated α3ß1 or α5ß1. Our results were the first evidence that, in the same tumor cells, different integrins can control various manifestations of tumor progression through distinct signaling pathways that are both common to various integrins and specific to a particular receptor.

New steroidal oxazolines, benzoxazoles and benzimidazoles related to abiraterone and galeterone.

Seven new oxazoline, benzoxazole and benzimidazole derivatives were synthesized from 3ß-acetoxyandrosta-5,16-dien-17-carboxylic, 3ß-acetoxyandrost-5-en-17ß-carboxylic and 3ß-acetoxypregn-5-en-21-oic acids. Docking to active site of human 17α-hydroxylase/17,20-lyase revealed that all oxazolines, as well as benzoxazoles and benzimidazoles comprising Δ16 could form stable complexes with enzyme, in which steroid moiety is positioned similarly to that of abiraterone and galeterone, and nitrogen atom coordinates heme iron, while 16,17-saturated benzoxazoles and benzimidazoles could only bind in a position where heterocycle is located nearly parallel to heme plane. Modeling of the interaction of new benzoxazole and benzimidazole derivatives with androgen receptor revealed the destabilization of helix 12, constituting activation function 2 (AF2) site, by mentioned compounds, similar to one induced by known antagonist galeterone. The synthesized compounds inhibited growth of prostate carcinoma LNCaP and PC-3 cells at 96 h incubation; the potency of 2'-(3ß-hydroxyandrosta-5,16-dien-17-yl)-4',5'-dihydro-1',3'-oxazole and 2'-(3ß-hydroxyandrosta-5,16-dien-17-yl)-benzimidazole was superior and could inspire further investigations of these compounds as potential anti-cancer agents.

Implication of integrin α2ß1 in anoikis of SK-Mel-147 human melanoma cells: a non-canonical function of Akt protein kinase.

Suppression of anoikis, a kind of apoptosis caused by disruption of contacts between cell and extracellular matrix, is an important prerequisite for cancer cell metastasis. In this communication, we demonstrate that shRNA-mediated depletion of α2 integrin subunit induces anoikis and substantially decreases colony-forming potential in SK-Mel-147 human melanoma cells. Suppression of α2ß1 upregulates the levels of pro-apoptotic protein p53 and cyclin-dependent kinase inhibitors p21 and p27. Concomitantly, we detected decrease in the levels of anti-apoptotic protein Bcl-2 and cell cycle regulator c-Myc. Moreover, depletion of α2ß1 reduces the activity of protein kinase Erk, while increases activity of Akt kinase. Pharmacological inhibition of P3IK kinase, an upstream activator of Akt, greatly enhanced anoikis in control cells while reduced that in cells with decreased levels of α2ß1. Of three isoforms of Akt, down-regulation of Akt1 greatly diminished anoikis of cells depleted of α2ß1, while down-regulation of Akt2 and Akt3 sharply increased anoikis in these cells. These findings were supported by the data of pharmacological inhibition of the Akt isoforms. Our results demonstrate for the first time that anoikis induced by α2ß1 integrin knockdown can be attenuated by Akt1 inhibition.

New azole derivatives of [17(20)E]-21-norpregnene: Synthesis and inhibition of prostate carcinoma cell growth.

A number of isoxazole, 1,2,3-triazole, tetrazole, and 1,2,4-oxadiazole derivatives of [17(20)E]-21-norpregnene comprising 3ß-hydroxy-5-ene and 3-oxo-4-ene fragments were prepared. Among the key steps for the synthesis of isoxazoles, 1,2,3-triazoles, and tetrazoles were (i) 1,3-dipolar cycloaddition of nitrile oxides or azides to acetylenes or nitriles and ii) dehydration of 17ß-hydroxy-17α-methylene-azoles to [17(20)E]-21-norpregnene derivatives. 1,2,4-Oxadiazoles were prepared through the formation of acetimidamides. Potency of the synthesized compounds to inhibit CYP17A1 and to suppress growth of prostate carcinoma cells was investigated. Among the new azole derivatives, four compounds were found possessing high anti-proliferative activity.

Conjugates of 17-substituted testosterone and epitestosterone with pyropheophorbide a differing in the length of linkers.

Conjugates of 17α-substituted testosterone (1 and 2) and 17ß-substituted epitestosterone (3 and 4) with pyropheophorbide a were synthesized. The scheme consisted of synthesis of 17α-hydroxy-3-oxopregn-4-en-21-oic and 17ß-hydroxy-3-oxopregn-4-en-21-oic acids, and their coupling with pyropheophorbide a by means of either ethylene diamine, or 1,5-diamino pentane linkers. Mutual influence of steroidal and macrocyclic fragments in conjugates molecules was dependent on configuration of C17 and length of linker, that was established by analysis of 1H NMR spectra and molecular models of conjugates. Studies of interaction of conjugates with prostate carcinoma cells revealed that their uptake and internalization were independent on the androgen receptor activity, but dependent on the structure of conjugates, decreasing in the following row: 3 > 4 ≥ 1 > 2. Conjugates significantly decreased the LNCaP and PC-3 cells growth at 96 h incubation. Epitestosterone derivatives 3 and 4 also showed superior anti-proliferative activity versus testosterone ones. Conformationally more rigid conjugates 1 and 3, comprising short linkers, were more active than those with long linkers; conjugate 3 was the most potent.

FOXQ1 controls the induced differentiation of melanocytic cells.

Oncogenic transcription factor FOXQ1 has been implicated in promotion of multiple transformed phenotypes in carcinoma cells. Recently, we have characterized FOXQ1 as a melanoma tumor suppressor that acts via repression of N-cadherin gene, and invasion and metastasis. Here we report that FOXQ1 induces differentiation in normal and transformed melanocytic cells at least partially via direct transcriptional activation of MITF gene, melanocytic lineage-specific regulator of differentiation. Importantly, we demonstrate that pigmentation induced in cultured melanocytic cells and in mice by activation of cAMP/CREB1 pathway depends in large part on FOXQ1. Moreover, our data reveal that FOXQ1 acts as a critical mediator of BRAFV600E-dependent regulation of MITF levels, thus providing a novel link between two major signal transduction pathways controlling MITF and differentiation in melanocytic cells.

Comparison of [17(20)E]-21-Norpregnene oxazolinyl and benzoxazolyl derivatives as inhibitors of CYP17A1 activity and prostate carcinoma cells growth.

Four new 4,5-dihydro-1,3-oxazole, and four new benzo-[d]-oxazole derivatives of [17(20)E]-21-norpregnene, differing in the structure of steroid moiety, were synthesized and evaluated for their potency to inhibit 17α-hydroxylase/17,20-lyase (CYP17A1) activity. Among new compounds, the only oxazolinyl derivative comprising 5-oxo-4,5-seco-3-yn- moiety potently inhibited CYP17A1. Binding modes of the oxazolinyl derivatives of [17(20)E]-21-norpregnene were analyzed by molecular dynamics simulations, and model of alternate, water-bridged type II interaction was proposed for these compounds. Eight new compounds, together with two CYP17A1-inhibiting oxazolinyl derivatives synthesized earlier, abiraterone and galeterone were evaluated for their potency to inhibit prostate carcinoma PC-3 and LNCaP cells growth. Oxazolinyl and benzoxazolyl derivatives comprising 3ß-hydroxy-5-ene moieties potently inhibited prostate carcinoma cell growth; inhibitory potencies of 3-oxo-4-en- and 5-oxo-4,5-seco-3-yn- derivatives were significantly lower.

Integrin α5ß1 simultaneously controls EGFR-dependent proliferation and Akt-dependent pro-survival signaling in epidermoid carcinoma cells.

To delineate distinctive role of the components of α5ß1 integrin-EGFR axis in control of epidermoid carcinoma cell proliferation, we performed individual inhibition of α5ß1 and EGFR via genetic and phamacological methods, respectively. We demonstrated that pharmacological inhibition of epidermal growth factor receptor (EGFR) significantly affected proliferation of A431 human cells by inducing the G0/G1 cell cycle arrest, whereas shRNA-mediated depletion of α5 subunit of α5ß1 integrin led to a similar type of cell cycle arrest followed by significant apoptosis. Both treatments resulted in suppression of activated (phosphorylated) forms of focal adhesion kinase (FAK) and Erk. However, unlike EGFR inhibition, depletion of α5 led to substantial suppression of AKT activity. Accordingly, pharmacological inhibition of EGFR and AKT recapitulated detrimental effects caused by shRNA-mediated depletion of α5. Moreover, depletion of α5 led to a severe drop in the amounts of active EGFR. Thus, for the first time, we demonstrated that α5ß1 integrin simultaneously maintains pro-survival signaling via continuous activation of AKT and up-regulates proliferation via activation of EGFR.

Fucans, but not fucomannoglucuronans, determine the biological activities of sulfated polysaccharides from Laminaria saccharina brown seaweed.

Sulfated polysaccharides from Laminaria saccharina (new name: Saccharina latissima) brown seaweed show promising activity for the treatment of inflammation, thrombosis, and cancer; yet the molecular mechanisms underlying these properties remain poorly understood. The aim of this work was to characterize, using in vitro and in vivo strategies, the anti-inflammatory, anti-coagulant, anti-angiogenic, and anti-tumor activities of two main sulfated polysaccharide fractions obtained from L. saccharina: a) L.s.-1.0 fraction mainly consisting of O-sulfated mannoglucuronofucans and b) L.s.-1.25 fraction mainly composed of sulfated fucans. Both fractions inhibited leukocyte recruitment in a model of inflammation in rats, although L.s.-1.25 appeared to be more active than L.s.-1.0. Also, these fractions inhibited neutrophil adhesion to platelets under flow. Only fraction L.s.-1.25, but not L.s.-1.0, displayed anticoagulant activity as measured by the activated partial thromboplastin time. Investigation of these fractions in angiogenesis settings revealed that only L.s.-1.25 strongly inhibited fetal bovine serum (FBS) induced in vitro tubulogenesis. This effect correlated with a reduction in plasminogen activator inhibitor-1 (PAI-1) levels in L.s.-1.25-treated endothelial cells. Furthermore, only parent sulfated polysaccharides from L. saccharina (L.s.-P) and its fraction L.s.-1.25 were powerful inhibitors of basic fibroblast growth factor (bFGF) induced pathways. Consistently, the L.s.-1.25 fraction as well as L.s.-P successfully interfered with fibroblast binding to human bFGF. The incorporation of L.s.-P or L.s.-1.25, but not L.s.-1.0 into Matrigel plugs containing melanoma cells induced a significant reduction in hemoglobin content as well in the frequency of tumor-associated blood vessels. Moreover, i.p. administrations of L.s.-1.25, as well as L.s.-P, but not L.s.-1.0, resulted in a significant reduction of tumor growth when inoculated into syngeneic mice. Finally, L.s.-1.25 markedly inhibited breast cancer cell adhesion to human platelet-coated surfaces. Thus, sulfated fucans are mainly responsible for the anti-inflammatory, anticoagulant, antiangiogenic, and antitumor activities of sulfated polysaccharides from L. saccharina brown seaweed.

Synthesis and cytotoxicity evaluation of 22,23-oxygenated stigmastane derivatives.

Starting from (22E)-3alpha,5alpha-cyclo-6beta-methoxystigmast-22-ene eighteen derivatives of (22S,23S)-22,23-oxidostigmastane, (22R,23R)-22,23-oxidostigmastane, and (22R,23R)-22,23-dihydroxystigmastane were synthesized and screened for cytotoxicity in human hepatoma Hep G2 cells and human breast carcinoma MCF-7 cells using MTT assay. Four compounds of this series exhibited high cytotoxicity in both cells; three compounds were selectively toxic in MCF-7 cells, one compound was toxic in Hep G2 cells, rather than in MCF-7 cells; four compounds at low concentrations increased MTT test values over the control.

A comparative study of the anti-inflammatory, anticoagulant, antiangiogenic, and antiadhesive activities of nine different fucoidans from brown seaweeds.

The anti-inflammatory, antiangiogenic, anticoagulant, and antiadhesive properties of fucoidans obtained from nine species of brown algae were studied in order to examine the influence of fucoidan origin and composition on their biological activities. All fucoidans inhibited leucocyte recruitment in an inflammation model in rats, and neither the content of fucose and sulfate nor other structural features of their polysaccharide backbones significantly affected the efficacy of fucoidans in this model. In vitro evaluation of P-selectin-mediated neutrophil adhesion to platelets under flow conditions revealed that only polysaccharides from Laminaria saccharina, L. digitata, Fucus evanescens, F. serratus, F. distichus, F. spiralis, and Ascophyllum nodosum could serve as P-selectin inhibitors. All fucoidans, except that from Cladosiphon okamuranus carrying substantial levels of 2-O-alpha-D-glucuronopyranosyl branches in the linear (1-->3)-linked poly-alpha-fucopyranoside chain, exhibited anticoagulant activity as measured by activated partial thromboplastin time whereas only fucoidans from L. saccharina, L. digitata, F. serratus, F. distichus, and F. evanescens displayed strong antithrombin activity in a platelet aggregation test. The last fucoidans potently inhibited human umbilical vein endothelial cell (HUVEC) tubulogenesis in vitro and this property correlated with decreased levels of plasminogen-activator inhibitor-1 in HUVEC supernatants, suggesting a possible mechanism of fucoidan-induced inhibition of tubulogenesis. Finally, fucoidans from L. saccharina, L. digitata, F. serratus, F. distichus, and F. vesiculosus strongly blocked MDA-MB-231 breast carcinoma cell adhesion to platelets, an effect which might have critical implications in tumor metastasis. The data presented herein provide a new rationale for the development of potential drugs for thrombosis, inflammation, and tumor progression.

Novel side chain modified Delta8(14)-15-ketosterols.

Synthesis of five novel Delta8(14)-15-ketosterols comprising modified side chains starting from ergosterol is described. Ergosteryl acetate was converted into (22E)-3beta-acetoxy-5alpha-ergosta-8(14),22-dien-15-one through three stages in 32% overall yield; further transformations of the product obtained led to (22E)-3beta-hydroxy-5alpha-ergosta-8(14),22-dien-15-one, (22S,23S)-3beta-hydroxy-22,23-oxido-5alpha-ergost-8(14)-en-15-one, (22R,23R)-3beta-hydroxy-22,23-oxido-5alpha-ergost-8(14)-en-15-one, (22R,23R)-5alpha-ergost-8(14)-en-15-on-3beta,22,23-triol and (22R,23R)-3beta-hydroxy-22,23-isopropylidenedioxy-5alpha-ergost-8(14)-en-15-one. New Delta8(14)-15-ketosterols were evaluated for their cytotoxicity and effects on sterol biosynthesis in human hepatoma Hep G2 cells in comparison with known 3beta-hydroxy-5alpha-cholest-8(14)-en-15-one. Among the compounds tested, (22R,23R)-3beta-hydroxy-22,23-oxido-5alpha-ergost-8(14)-en-15-one was found to be the most potent inhibitor of sterol biosynthesis (IC(50)=0.6+/-0.2microM), whereas (22R,23R)-5alpha-ergost-8(14)-en-15-on-3beta,22,23-triol exhibited the highest cytotoxicity (TC(50)=12+/-3microM at a 24h incubation).