Daidzein, R-(+)equol and S-(-)equol inhibit the invasion of MDA-MB-231 breast cancer cells potentially via the down-regulation of matrix metalloproteinase-2

PURPOSE: Soy isoflavones may inhibit tumor cell invasion and metastasis via their effects on matrix metalloproteinases (MMPs) and their tissue inhibitors (TIMPs). The current study investigates the effects of daidzein, R- and S-equol on the invasion of MDA-MB-231 human breast cancer cells and the effects of these compounds on MMP/TIMP expression at the mRNA level. METHODS: The anti-invasive effects of daidzein, R- and S-equol (0, 2.5, 10, 50 μM) on MDA-MB-231 cells were determined using the Matrigel invasion assay following 48-h exposure. Effects on MMP-2, MMP-9, TIMP-1 and TIMP-2 expression were assessed using real-time PCR. Chiral HPLC analysis was used to determine intracellular concentrations of R- and S-equol. RESULTS: The invasive capacity of MDA-MB-231 cells was significantly reduced (by approximately 50-60 %) following treatment with 50 μM daidzein, R- or S-equol. Anti-invasive effects were also observed with R-equol at 2.5 and 10 μM though overall equipotent effects were induced by all compounds. Inhibition of invasion induced by all three compounds at 50 μM was associated with the down-regulation of MMP-2, while none of the compounds tested significantly affected the expression levels of MMP-9, TIMP-1 or TIMP-2 at this concentration. Following exposure to media containing 50 μM R- or S-equol for 48-h intracellular concentrations of R- and S-equol were 4.38 ± 1.17 and 3.22 ± 0.47 nM, respectively. CONCLUSION: Daidzein, R- and S-equol inhibit the invasion of MDA-MB-231 human breast cancer cells in part via the down-regulation of MMP-2 expression, with equipotent effects observed for the parent isoflavone daidzein and the equol enantiomers.

Immune Responses and Therapeutic Antitumor Effects of an Experimental DNA Vaccine Encoding Human Papillomavirus Type 16 Oncoproteins Genetically Fused to Herpesvirus Glycoprotein D

Recombinant adenovirus or DNA vaccines encoding herpes simplex virus type 1 (HSV-1) glycoprotein D (gD) genetically fused to human papillomavirus type 16 (HPV-16) oncoproteins (E5, E6, and E7) induce antigen-specific CD8(+) T-cell responses and confer preventive resistance to transplantable murine tumor cells (TC-1 cells). In the present report, we characterized some previously uncovered aspects concerning the induction of CD8(+) T-cell responses and the therapeutic anticancer effects achieved in C57BL/6 mice immunized with pgD-E7E6E5 previously challenged with TC-1 cells. Concerning the characterization of the immune responses elicited in mice vaccinated with pgD-E7E6E5, we determined the effect of the CD4(+) T-cell requirement, longevity, and dose-dependent activation on the E7-specific CD8(+) T-cell responses. In addition, we determined the priming/boosting properties of pgD-E7E6E5 when used in combination with a recombinant serotype 68 adenovirus (AdC68) vector encoding the same chimeric antigen. Mice challenged with TC-1 cells and then immunized with three doses of pgD-E7E6E5 elicited CD8(+) T-cell responses, measured by intracellular gamma interferon (IFN-gamma) and CD107a accumulation, to the three HPV-16 oncoproteins and displayed in vivo antigen-specific cytolytic activity, as demonstrated with carboxyfluorescein diacetate succinimidyl ester (CFSE)-labeled target cells pulsed with oligopeptides corresponding to the H-2D(b)-restricted immunodominant epitopes of the E7, E6, or E5 oncoprotein. Up to 70% of the mice challenged with 5 x 10(5) TC-1 cells and immunized with pgD-E7E6E5 controlled tumor development even after 3 days of tumor cell challenge. In addition, coadministration of pgD-E7E6E5 with DNA vectors encoding pGM-CSF or interleukin-12 (IL-12) enhanced the therapeutic antitumor effects for all mice challenged with TC-1 cells. In conclusion, the present results expand our previous knowledge on the immune modulation properties of the pgD-E7E6E5 vector and demonstrate, for the first time, the strong antitumor effects of the DNA vaccine, raising promising perspectives regarding the development of immunotherapeutic reagents for the control of HPV-16-associated tumors.

Gamma-linolenic Acid Alters Ku80, E2F1, and Bax Expression and Induces Micronucleus Formation in C6 Glioma Cells In Vitro

Gamma-linolenic acid (GLA) is an inhibitor of tumor cell proliferation in both in vitro and in vivo conditions. The aim of this study was to investigate the effects of 150 mu M GLA on the expression of E2F1, cyclin D1, bax, bcl2, Ku70, and Ku80 in C6 rat glioma cells. The Ku proteins were chosen as previous studies have shown that loss or reduction in their expression causes increased DNA damage and micronucleus formation in the presence of radiation. The fact that GLA exposure is known to enhance the efficacy of radiation treatment raised the question whether the Ku proteins could be involved in this effect as seen for other molecules such as roscovitine and flavopiridol. GLA altered the mRNA expression of E2F1, cyclin D1, and bax, but no changes were found for bcl2, Ku70, and Ku80. Alterations in protein expression were observed for bax, Ku80, and E2F1. The 45% decrease in E2F1 expression was proportional to decreased cell proliferation (44%). Morphological analysis found a 25% decrease in mitotic activity in the GLA-treated cells, which was accompanied by a 49% decrease in S-phase by FACS analysis. A 39% increase in the number of micronuclei detected by Hoechst fluorescence points to GLA`s effects on cell division even at concentrations that do not produce significant increases in apoptosis. Most important was the finding that Ku80 expression, a critical protein involved in DNA repair as a heterodimer with Ku70, was decreased by 71%. It is probable that reduced Ku80 is responsible for the increase in micronucleus formation in GLA-treated cells in a similar manner to that found in Ku80 null cells exposed to radiation. The decreased expression of Ku80 and E2F1 could make cells more susceptible to radiotherapy and chemotherapy. (C) 2009 IUBMB

Synthesis, characterization, X-ray structure and in vitro anti mycobacterial and antitumoral activities of Ru(II) phosphine/diimine complexes containing the ""SpymMe(2)"" ligand, SpymMe(2)=4,6-dimethyl-2-mercaptopyrimidine

The reaction of cis-[RuCl2(dppb)(N-N)], dppb = 1,4-bis(diphenylphosphino)butane, complexes with the ligand HSpymMe(2), 4,6-dimethyl-2-mercaptopyrimidine, yielded the cationic complexes [Ru(SpymMe(2))(dppb)(N-N)]PF6, N-N = bipy (1) and Me-bipy (2), bipy = 2,2`-bipyridine and Me-bipy = 4,4`dimethyl-2,2`-bipyridine, which were characterized by spectroscopic and electrochemical techniques and X-ray crystallography and elemental analysis. Additionally, preliminary in vitro tests for antimycobacterial activity against Mycobacterium tuberculosis H37Rv ATCC 27264 and antitumor activity against the MDA-MB-231 human breast tumor cell line were carried out on the new complexes and also on the precursors cis-[RuCl2(dppb)(N-N)], N-N = bipy (3) and Me-bipy (4) and the free ligands dppb, bipy, Me-bipy and SpymMe(2). The minimal inhibitory concentration (MIC) of compounds needed to kill 90% of mycobacterial cells and the IC50 values for the antitumor activity were determined. Compounds 1-4 exhibited good in vitro activity against M. tuberculosis, with MIC values ranging between 0.78 and 6.25 mu g/mL, compared to the free ligands (MIC of 25 to >50 mu g/mL) and the drugs used to treat tuberculosis. Complexes I and 2 also showed promising antitumor activity, with IC50 values of 0.46 +/- 0.02 and 0.43 +/- 0.08 mu M, respectively, against MDA-MB-231 breast tumor cells. (C) 2008 Elsevier Inc. All rights reserved.

A new nitrosyl ruthenium complex: Synthesis, chemical characterization, in vitro and in vivo antitumor activities and probable mechanism of action

This study describes the synthesis of a new ruthenium nitrosyl complex with the formula [RuCl(2)NO(BPA)] [BPA = (2-hydroxybenzyl)(2-methylpyridyl)amine ion], which was synthesized and characterized by spectroscopy, cyclic voltammetry, X-ray crystallography, and theoretical calculation data. The biological studies of this complex included in vitro cytotoxic assays, which revealed its activity against two different tumor cell lines (HeLa and Tm5), with efficacy comparable to that of cisplatin, a metal-based drug that is administered in clinical treatment. The in vivo studies showed that [RuCl2NO(BPA)] is effective in reducing tumor mass. Also, our results suggest that the mechanism of action of [RuCl(2)NO(BPA)] includes binding to DNA, causing fragmentation of this biological molecule, which leads to apoptosis. (C) 2011 Elsevier Masson SAS. All rights reserved.

(+)- and (-)-Mutisianthol: First Total Synthesis, Absolute Configuration, and Antitumor Activity

The first synthesis of the natural product (+)-mutisianthol was accomplished in 11 steps and in 21% overall yield from 2-methylanisole. The synthesis of its enantiomer was also performed in a similar overall yield. The absolute configuration of the sesquiterpene (+)-mutisianthol was assigned as (1S,3R). Key steps in the route are the asymmetric hydrogenation of a nonfunctionalized olefin using chiral iridium catalysts and the ring contraction of 1,2-dihydronaphthalenes using thallium(III) or iodine(III). The target molecules show moderate activity against the human tumor cell lines SF-295, HCT-8, and MDA-MB-435.

Double stranded DNA-binding studies of potential Rhodium(ll) antitumor complexes

In 1952, Dwyer and coworkers began testing a series of metal complexes for potential inhibition of cancer cell proliferation in animals.[l] The complexes tested were unsuitable for such studies due to their high toxicity. Therefore, no further work was done on the project. However, in 1965, Rosenberg and coworkers revisited the possibility of potential metal-based drugs. Serendipitously, they discovered that cis-diamminedichloroplatinum(lI) (cisplatin) inhibits cell division in E. coli.[2] Further studies of this and other platinum compounds revealed inhibition of tumor cell lines sarcoma 180 and leukemia LI2l0 in mice.[l] Cisplatin was approved by the Food and Drug Administration in 1970 as a chemical chemotherapeutic agent in the treatment of cancer. The drug has primarily been used in the treatment of testicular and ovarian cancers, although the powerful chemotherapeutic properties of the compound indicate use against a variety of other cancers.[3] The toxicity of this compound, however, warrants the development of other metal-based potential antitumor agents. The success of cisplatin, a transition-metal-based chemotherapeutic, opened the doors to a host of research on the antitumor effects of other transition-metal complexes. Beginning in the 1970s, researchers looked to rhodium for potential use in antitumor complexes. Dirhodium complexes with bridging equatorial ligands (Figure I) were the primary focus for this research. The overwhelming majority of these complexes were dirhodium(II) carboxylate complexes, containing two rhodium(II) centers, four equatorial ligands in a lantero formation around the metal center, and an axial ligand on either end. The family of complexes in Figure 1 will be referred to as dirhodium(II) carboxylate complexes. The dirhodium centers are each d? with a metal-metal bond between them. Although d? atoms are paramagnetic, the two unpaired electrons pair to make the complex diamagnetic. The basic formula of the dirhodium(lI) carboxylate complexes is Rh?(RCOO)?(L)? with R being methyl, ethyl, propyl, or butyl groups and L being water or the solvent in which the complex was crystalized. Of these dirbodium(II) carboxylate complexes, our research focuses on Rb la and two other similar complexes Rh2 and Rh3 (Figure 2). Rh2 is an activated form of Rhla, with four acetonitrile groups in place of two of the bidentate acetate ligands. Rh3 is similar to Rhla, with trifluoromethyl groups in place of the methyl groups on the acetate ligands.

Aspectos estruturais, farmacológicos e biológicos de fucanas da alga marrom sargassum vulgare

The present study examines the chemical composition and their effects on free radicals, inflammation, angiogenesis, coagulation, VEGF effects and cellular proliferation of a polysaccharides from alga Sargassum vulgare. The sulfated polysaccharide was extracted from brown seaweed by proteolysis with enzymes maxataze. The presence of proteins and sugars were observed in crude polysaccharides. Fractionation of this crude extract was made with growing concentration of acetone (0.3-1.5 v) and produced four groups of polysaccharides. Anionic polysaccharides from brown seaweed Sargassum vulgare, SV1and PSV1 were fractionated (SV1) and purified (PSV1), and displayed with high total sugars and sulfate content and very low level of protein. This fucan SV1 contains low levels of protein and high carbohydrate and sulfate content. This polysaccharides prolonged activated partial thromboplastin time (aPTT) at 50 μg (>240 s). SV1 was found to have no effect on prothrombin time (PT), corresponding to the extrinsic pathway of coagulation. SV1 exhibits high antithrombotic action in vivo, with a concentration ten times higher than heparin. Polysaccharides from S. vulgare promoted direct inhibition enzymatic activity of thrombin and stimulated enzymatic activity of FXa. SV1 showed optimal inhibitory activity of thrombin (50.2±0.28%) at a concentration of 25 μg/mL. Its antioxidant action on scavenging radicals by DPPH was (22%), indicating the polymer has no cytotoxic action (hemolytic) on ABO and Rh blood types in different erythrocyte groups and displays strong anti-inflammatory action on all concentrations tested in the carrageenan-induced paw edema model, demonstrated by reduced edema and cellular infiltration. Angiogenesis is a dynamic process of proliferation and differentiation. It requires endothelial proliferation, migration, and tube formation. In this context, endothelial cells are a preferred target for several studies and therapies. The antiangiogenic efficacy of polysaccharides was examined in vivo in the chick chorioallantoic membrane (CAM) model by using fertilized eggs. Decreases in the density of the capillaries were assessed and scored. The results showed that SV1 and PSV1 have an inhibitory effect on angiogenesis. These results were also confirmed by inhibition tubulogenesis in rabbit aorta endothelial cell (RAEC) in matrigel. These compounds were assessed in Apoptosis assay (Annexin V - FITC / PI) and cell viability by MTT assay of RAEC. These polysaccharides do not affect the viability and do not have apoptotic or necrotic action. RAEC cell when incubated with SV1 and PSV1showed inhibition of VEGF secretion, observed when compounds were incubated at 25, 50 and 100 μg/μL. The VEGF secretion with the RAEC cell line for 24 h, was more effective for PSV1 at 50 μg/μL(71.4%) than SV1 100 μg/μL (75.9%). SV1 and PSV1 had an antiproliferative action (47%) against tumor cell line HeLa. Our results indicate that these sulfated polysaccharides have antiangiogenic and antitumoral actions

Avaliação das atividades antioxidante, anticoagulante e antiproliferativa de extratos aquosos de marsdenia megalantha

The species of the genus Marsdenia, Apocynaceae, are widely used in folk medicine of several countries. In Brazil is found several species belonging to this genus. The in vitro antioxidant, anticoagulant and antiproliferative activities were evaluated to aqueous extracts of stalk, leaf and root of Marsdenia megalantha. In the total antioxidant capacity assay (expressed as ascorbic acid equivalents) the stalk extract showed 76.0 mg/g, while leaf and root extracts 141.3 mg/g and 57.0 mg/g, respectively. The stalk and leaf extracts showed chelating activity around 40% at 1.5 mg/mL, while root extract, at the same concentration showed, 17%. Only the leaf extract showed a significant ability in superoxide scavenging (80% at 0.8 mg/mL). Any extract was able in scavenge hydroxyl, as well anticoagulant activity. The antiproliferative activity of the extracts was evaluated against HeLa tumor cell line. The extracts inhibited in a dose-dependent manner the cell growth. However, the leaf extract showed 80% of inhibition at 1.0 mg/mL, while stalk and root extracts inhibited 63% and 30%, respectively. To assess the mechanism of cell death caused by the leaf extract in HeLa, was performed flow cytometry and western blot. The results show that leaf extract induces cell death by apoptosis through an activation caspase-independent pathway. These data indicate that stalk and leaf extracts obtained have potential to be used as antioxidants and anticancer drugs

Avaliação do efeito do composto tipo heparina isolado do caranguejo Chaceon fenneri na hemostasia e na morte celular

Heparin is a pharmaceutical animal widely used in medicine due to its potent anticoagulant effect. Furthermore, it has the ability to inhibit the proliferation, invasion and adhesion of cancer cells to vascular endothelium. However, its clinical applicability can be compromised by side effects such as bleeding. Thus, the search for natural compounds with low bleeding risk and possible therapeutic applicability has been targeted by several research groups. From this perspective, this study aims to evaluate the hemorrhagic and anticoagulant activities and citotoxic effect for different tumor cell lines (HeLa, B16-F10, HepG2, HS-5,) and fibroblast cells (3T3) of the Heparin-like from the crab Chaceon fenneri (HEP-like). The HEP-like was purified after proteolysis, ion-exchange chromatography, fractionation with acetone and characterized by electrophoresis (agarose gel) and enzymatic degradation. Hep-like showed eletroforetic behavior similar to mammalian heparin, and high trisulfated /Nacetylated disaccharides ratio. In addition, HEP-like presented low in vitro anticoagulant activity using aPTT and a minor hemorrhagic effect when compared to mammalian heparin. Furthermore, the HEP-like showed significant cytotoxic effect (p<0.001) on HeLa, HepG2 and B16-F10 tumor cells with IC50 values of 1000 ug/mL, after incubation for 72 hours. To assess the influence of heparin-like on the cell cycle in HeLa cells, analysis was performed by flow cytometry. The results of this analysis showed that HEP-like influence on the cell cycle increasing S phase and decreasing phase G2. Thus, these properties of HEP-like make these compounds potential therapeutic agents

Bioprospecção de extratos metanólicos de macroalgas marinhas do litoral do Rio Grande do Norte: avaliação das atividades anticoagulante, antioxidante e antiproliferativa

Seaweeds are organisms known to exhibit a variety of biomolecules with pharmacological properties. The coast of Rio Grande do Norte has over 100 species of seaweeds, most of them not yet explored for their pharmacological potential. Sugars and phenolic compounds are the most studied of these being assigned a range of biological properties, such as anticoagulant , antiinflammatory, antitumor and antioxidant activities. In this work, we obtained methanolic extracts from thirteen seaweeds of the coast of Rio Grande do Norte (Dictyota cervicornis; Dictiopterys delicatula; Dictyota menstruallis; D. mertensis; Sargassum filipendula; Spatoglossum schröederi; Acanthophora specifera; Botryocladia occidentalis; Caulerpa cupresoides; C. racemosa; C. prolifera; C. sertularioides e Codium isthmocladum). They were evaluated as anticoagulant and antioxidant drugs, as well as antiproliferative drugs against the tumor cell line HeLa. None of the methanolic extracts showed anticoagulant activity, but when they were evaluated as antioxidant drugs all of extracts showed antioxidant activity in all tests performed (total antioxidant capacity, sequestration of superoxide and hydroxyl radicals, ferric chelation and reductase activity), especially the algae D. mentrualis, D. cilliolata and C. prolifera, who had the greatest potential to donate electrons.In addition, the ability of iron ions chelation appears as the main antioxidant mechanism of the methanolic extracts of these seaweeds mainly for the extract of the C. racemosa seaweed, which reached almost 100% activity. In the MTT assay, all extracts showed inhibitory activity at different levels againts HeLa cells. Moreover, D. cilliolata (MEDC) and D. menstrualis (MEDM) extracts showed specific activity to this cell line, not inhibiting the viability of 3T3 normal cell line, so they were chosen for detailing the antiproliferative mechanism of action. Using flow cytometry, fluorescence microscopy and in vitro assays we demonstrated that MEDC and MEDM induced apoptosis in HeLa cells by activation of caspases 3 and 9 and yet, MEDC induces cell cycle arrest in S phase. Together, these results showed that the methanolic extracts of brown seaweed D. menstrualis and D. cilliolata may contain agents with potential use in combatting cells from human uterine adenocarcinoma. This study also points to the need for more in-depth research on phytochemical and biological context to enable the purification of biologically active products of these extracts

Identificação e avaliação de propriedades de polissacarídeos sulfatados de diferentes fontes naturais que possibilitem sua aplicabilidade biotecnológica

Sulfated polysaccharides (SP) are widely distributed in animals and seaweeds tissues. These polymers have been studied in light of their important pharmacological activities, such as anticoagulant, antioxidant, antitumoral, anti-inflammatory, and antiviral properties. On other hand, SP potential to synthesize biomaterials like as nanoparticules has not yet been explored. In addition, to date, SP have only been found in six plants and all inhabit saline environments. However, the SP pharmacological plant activities have not been carrying out. Furthermore, there are no reports of SP in freshwater plants. Thus, do SP from marine plants show pharmacological activity? Do freshwater plants actually synthesize SP? Is it possible to synthesize nanoparticles using SP from seaweed? In order to understand this question, this Thesis was divided into tree chapters. In the first chapter a sulfated polysaccharide (SPSG) was successfully isolated from marine plant Halodule wrightii. The data presented here showed that the SPSG is a 11 kDa sulfated heterogalactan contains glucose and xylose. Several assays suggested that the SPSG possessed remarkable antioxidant properties in different in vitro assays and an outstanding anticoagulant activity 2.5-fold higher than that of heparin Clexane® in the aPTT test; in the next chapter using different tools such as chemical and histological analyses, energy-dispersive X-ray analysis (EDXA), gel electrophoresis and infra-red spectroscopy we confirm the presence of sulfated polysaccharides in freshwater plants for the first time. Moreover, we also demonstrate that SP extracted from E. crassipes root has potential as an anticoagulant compound; and in last chapter a fucan, a sulfated polysaccharide, extracted from the brown seaweed was chemically modified by grafting hexadecylamine to the polymer hydrophilic backbone. The resulting modified material (SNFuc) formed nanosized particles. The degree of substitution for hydrophobic chains of 1H NMR was approximately 93%. SNFfuc-TBa125 in aqueous media had a mean diameter of 123 nm and zeta potential of -38.3 ± 0.74 mV, measured bydynamic light scattering. Tumor-cell (HepG2, 786, H-S5) proliferation was inhibited by 2.0 43.7% at SNFuc concentrations of 0.05 0.5 mg/ mL and RAEC non-tumor cell line proliferation displayed inhibition of 8.0 22.0%. On the other hand, nanogel improved CHO and RAW non-tumor cell line proliferation in the same concentration range. Flow cytometric analysis revealed that this fucan nanogel inhibited 786 cell proliferation through caspase and caspaseindependent mechanisms. In addition, SNFuc blocks 786 cell passages in the S and G2-M phases of the cell cycle

Antitumor and antimycobacterial activities of cyclopalladated complexes: X-ray structure of [Pd(C-2,N-dmba)(Br)(tu)] (dmba = N,N-dimethylbenzylamine, tu = thiourea)

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)

Antitumoral, mutagenic and (anti)estrogenic activities of tingenone and pristimerin

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)

Assessment of estrogenic, mutagenic and antimutagenic activity of nemorosone

Currently, a wide range of research involving natural products is focused on the discovery of new drugs in many different therapeutic areas. A great number of the synthetic compounds on the market were derived from natural products, especially plants. Nemorosone is the major constituent of the floral resin of Clusia rosea Jacq., Clusiaceae, and in Cuban propolis. In vitro studies have shown cytotoxic activity in this substance against various tumor cell lines, including those resistant to various cytotoxic drugs, whereas it has low cytotoxicity to non-tumoral cells. Therefore, in order to characterize the biological activity of nemorosone, a substance with potential antitumor activity, and in view of preclinical testing of the toxicity of drug candidate compounds, the main aim of this study was to determine the mutagenic and antimutagenic activity of nemorosone by the Ames test, using the strains TA97a, TA98, TA100 and TA102 of Salmonella typhimurium. Secondly, to characterize the estrogenic activity in an experimental recombinant yeast model (Recombinant Yeast Assay) mutagenic activity was observed at in any of the concentrations in any of the test strains. To evaluate the antimutagenic potential, direct and indirect mutagenic agents were used: 4 nitro-o-phenylenediamine (NPD), mitomycin C (MMC) and aflatoxin B1 (AFL). Nemorosone showed moderate antimutagenic activity (inhibition level 31%), in strain TA100 in the presence of AFL, and strong antimutagenic activity in TA102 against MMC (inhibition level 53%). Estrogenic activity was observed, with an EEq of 0.41±0.16 nM at various tested concentrations.