Structure Elucidation, Antimicrobial and Cytotoxic Activities of a Halimane Isolated from Vellozia kolbekii Alves (Velloziaceae).

A new halimane diterpene was isolated from Vellozia kolbekii Alves (Velloziaceae) and identified as (5R,8R,9S,13R)-halim-1,10-ene-15,16-diol (1). It showed cytotoxicity against three human cancer cell lines, SF-295 (glioblastoma), MDA-MB-435 (melanoma), and HCT-8 (colon adenocarcinoma). In the mechanism of cytotoxic action, halimane 1 interferes in two major phases of the cell cycle: in S phase, in which DNA synthesis occurs and where it is very sensitive to damage, and G2M phase which is the phase of preparation for mitosis and mitosis itself, showing apoptosis-inducing properties. Antimicrobial activity towards Gram-positive and Gram-negative bacteria was studied and, against Bacillus cereus, B. subtilis, Escherichia coli, and Pseudomonas aeruginosa, a MIC value of 0.025 µM was observed for halimane 1, which is more active than the positive control chloramphenicol.

Design, synthesis and biological evaluation of (E)-2-(2-arylhydrazinyl)quinoxalines, a promising and potent new class of anticancer agents.

A series of forty-seven quinoxaline derivatives, 2-(XYZC6H2CHN-NH)-quinoxalines, 1, have been synthesized and evaluated for their activity against four cancer cell lines: potent cytotoxicities were found (IC50 ranging from 0.316 to 15.749 µM). The structure-activity relationship (SAR) analysis indicated that the number, the positions and the type of substituents attached to the aromatic ring are critical for biological activity. The activities do not depend on the electronic effects of the substituents nor on the lypophilicities of the molecules. A common feature of active compounds is an ortho-hydroxy group in the phenyl ring. A potential role of these ortho-hydroxy derivatives is as N,N,O-tridentate ligands complexing with a vital metal, such as iron, and thereby preventing proliferation of cells. The most active compound was (1: X,Y=2,3-(OH)2, Z=H), which displayed a potent cytotoxicity comparable to that of the reference drug doxorubicin.

Goat milk with and without increased concentrations of lysozyme improves repair of intestinal cell damage induced by enteroaggregative Escherichia coli.

BACKGROUND: Enteroaggregative Escherichia coli (EAEC) causes diarrhea, malnutrition and poor growth in children. Human breast milk decreases disease-causing bacteria by supplying nutrients and antimicrobial factors such as lysozyme. Goat milk with and without human lysozyme (HLZ) may improve the repair of intestinal barrier function damage induced by EAEC. This work investigates the effect of the milks on intestinal barrier function repair, bacterial adherence in Caco-2 and HEp-2 cells, intestinal cell proliferation, migration, viability and apoptosis in IEC-6 cells in the absence or presence of EAEC. METHODS: Rat intestinal epithelial cells (IEC-6, ATCC, Rockville, MD) were used for proliferation, migration and viability assays and human colon adenocarcinoma (Caco-2, ATCC, Rockville, MD) and human larynx carcinoma (HEp-2, ATCC, Rockville, MD) cells were used for bacterial adhesion assays. Goats expressing HLZ in their milk were generated and express HLZ in milk at concentration of 270 µg/ml. Cells were incubated with pasteurized milk from either transgenic goats expressing HLZ or non-transgenic control goats in the presence and absence of EAEC strain 042 (O44:H18). RESULTS: Cellular proliferation was significantly greater in the presence of both HLZ transgenic and control goat milk compared to cells with no milk. Cellular migration was significantly decreased in the presence of EAEC alone but was restored in the presence of milk. Milk from HLZ transgenic goats had significantly more migration compared to control milk. Both milks significantly reduced EAEC adhesion to Caco-2 cells and transgenic milk resulted in less colonization than control milk using a HEp-2 assay. Both milks had significantly increased cellular viability as well as less apoptosis in both the absence and presence of EAEC. CONCLUSIONS: These data demonstrated that goat milk is able to repair intestinal barrier function damage induced by EAEC and that goat milk with a higher concentration of lysozyme offers additional protection.

Cytotoxic activity of fungal strains isolated from the ascidian Eudistoma vannamei.

The cytotoxic activity at 50 µg/ml of extracts obtained from eleven fungal strains associated to Eudistoma vannamei, an endemic ascidian from Northeast Brazil, against two cell lines, i.e., the HCT-8 (colon cancer) and the MDA-MB-435 (melanoma) cell lines, was investigated. The most promising extract (EV10) was obtained from a fungus identified as Aspergillus sp. by molecular analysis and was selected for bioassay-guided isolation of its active principals. Large-scale fermentation of EV10 in potato-dextrose broth followed by chromatographic purification of the active extract from the liquid medium allowed the isolation of the isocoumarins mellein, cis-4-hydroxymellein, and trans-4-hydroxymellein, besides penicillic acid. All isolated compounds were tested for their cytotoxicity against the tumor cell lines MDA-MB-435 and HCT-8 and revealed penicillic acid as the only cytotoxic compound (cell growth inhibitions >95%).

Studies on the secondary metabolites of a Pseudoalteromonas sp. isolated from sediments collected at the northeastern coast of Brazil.

Continuing search for anticancer compounds from the marine environment, we have studied microorganisms that inhabit intertidal sediments of the northeastern Brazilian coast. Of the 32 strains isolated, 13 were selected for biological evaluation of their crude extracts. The acetate extract obtained from a Gram-negative bacterium was strongly active against cancer cell lines with IC(50) values that ranged from 0.04 (HL60 leukemia cells) to 0.26 µg/ml (MDA MB-435 melanoma cells). The bacterium was identified as a Pseudoalteromonas sp. based on 16S rRNA gene sequencing. A bioassay-guided fractionation of the active extract led to the isolation of prodigiosin, a well-known tripyrrole red pigment with immunosuppressive and anticancer activities. Further experiments with ErbB-2 overexpressing cell lines, including HB4a-C3.6 (moderate overexpression), HB4a-C5.2 (high overexpression), and the parental HB4a cell line, were performed. Prodigiosin was moderately active toward HB4a cells with an IC(50) of 4.6 µg/ml, while it was 115 and 18 times more active toward HB4a-C3.6 cells (IC(50) of 0.04 µg/ml) and HB4a-C5.2 (IC(50) of 0.26 µg/ml) cells, respectively. These data suggest that, in spite of its previously described apoptosis-inducing properties, prodigiosin can selectively recognize cells overexpressing ErbB-2, which could be highly appealing in human breast cancer therapy.

Involvement of intrinsic mitochondrial pathway in neosergeolide-induced apoptosis of human HL-60 leukemia cells: the role of mitochondrial permeability transition pore and DNA damage.

CONTEXT: Quassinoids are biologically active secondary metabolites found exclusively in the Simaroubaceae family of plants. These compounds generally present important biological properties, including cytotoxic and antitumor properties. OBJECTIVE: In the present study, the cytotoxic effects of neosergeolide, a quassinoid isolated from Picrolemma sprucei Hook. f., were evaluated in human promyelocytic leukemia cells (HL-60). MATERIALS AND METHODS: Cytotoxicity and antiproliferative effects were evaluated by the MTT assay, May-Grünwald-Giemsa's staining, BrdU incorporation test, and flow cytometry procedures. The comet assay and micronuclei analysis were applied to determine the genotoxic and mutagenic potential of neosergeolide. RESULTS: After 24 h exposure, neosergeolide strongly inhibited cancer cell proliferation (IC50 0.1 µM), and its activity seemed to be selective to tumor cells because it had no antiproliferative effect on human peripheral blood mononuclear cells (PBMC) at tested concentrations. Apoptosis was induced at submicromolar concentrations (0.05, 0.1, and 0.2 µM) as evidenced by morphological changes, mitochondrial depolarization, phosphatidylserine externalization, caspases activation, and internucleosomal DNA fragmentation. Additionally, neosergeolide effects were prevented by cyclosporine A (CsA), an inhibitor of the mitochondrial permeability transition (MPT) pore, which reinforced the participation of intrinsic pathways in the apoptotic process induced by this natural quassinoid. Direct DNA damage was further confirmed by comet assay and cytokinesis-block micronucleus test. DISCUSSION AND CONCLUSION: The present study provided experimental evidence to support the underlying mechanism of action involved in the neosergeolide-mediated apoptosis. In addition, no antiproliferative effect or DNA damage effect of neosergeolide was evident in PBMC, highlighting its therapeutic potential.

In vitro studies on Bothrops venoms cytotoxic effect on tumor cells.

BACKGROUND: Animal venoms are complex mixtures of proteins and non proteins components with several biological activities. Snake venoms represent an essentially unexplored source of bioactive compounds that may cure disease conditions which do not respond to currently available therapies. These venoms possess many pharmacological activities, as cytotoxic and/or lytic effects on tumor cells in vitro. Herein, were investigated the in vitro cytotoxicity of three Bothrops venoms in tumor cell lines. METHODS: Cytotoxic effect was evaluated in HCT-8 (colon - human), SF-295 (nervous system - human), HL-60 (human leukemia) and MDAMB-435 (breast - human). Cell density and membrane integrity were determined by the exclusion of propidium iodide. To determine whether Bothrops venoms treated cells were undergoing an apoptotic and/ or necrosis death, phosphatidylserine (PS) externalization was measured after the incubation with the venom. RESULTS: Botrhops venons showed significant cytotoxcity against all cell lines in study. Cell density and membrane integrity were determined by the exclusion of propidium iodide. The Bothrops venoms reduced the cell number and revealed the presence of a necrotic population when the cells was exposed to the B. pauloensis B. diporus and B. pirajai venoms. To determine whether Bothrops venoms treated cells were undergoing an apoptotic and/or necrosis death, PS externalization was measured after the incubation with the venom and it was observed necrotic and apoptotic cells. CONCLUSIONS: All Bothrops venoms tested showed cytotoxicity against tumor cell lines through inducing of necrosis and apoptosis.

Biological evaluation of isoniazid derivatives as an anticancer class.

A series of thirty-two isoniazid derivatives have been evaluated for their activity against four human cancer cell lines with potent cytotoxicity (IC50 ranging from 0.61 to 3.36 µg/mL). The structure-activity relationship (SAR) analysis indicated the number, the positions, and the types of substituents attached to the aromatic ring as being critical factors for the biological activity. Briefly, we observed that the presence of a hydroxyl group on the benzene ring plays an important role in the anticancer activity of this series, especially when it is located in ortho-position. Among the thirty-two compounds, three displayed good cytotoxic activity when compared to the reference drug doxorubicin and are thus being considered leading compounds of this new class.

Mefloquine-oxazolidine derivatives: a new class of anticancer agents.

A series of 23 racemic mefloquine-oxazolidine derivatives, 4-[3-(aryl)hexahydro[1,3]oxazolo[3,4-a]pyridin-1-yl]-2,8-bis(trifluoromethyl)quinolines, derived from (R*, S*)-(±)-mefloquine and arenealdehydes, have been evaluated for their activity against four cancer cell lines (HCT-8, OVCAR-8, HL-60, and SF-295). Good cytotoxicities have been determined with IC50 values ranging from 0.59 to 4.79 µg/mL. In general compounds with aryl groups having strong electron-releasing substituents, such as HO and MeO, or electron-rich heteroaryl groups, for example imidazol-2-y-l, are active. However, other factors such as steric effects may play a role. As both the active and non-active conformations of the mefloquine-oxazolidine derivatives are similar, it is concluded that molecular conformations do not play a significant role either. This study is the first to evaluate mefloquine derivatives as antitumor agents. The mefloquine-oxazolidine derivatives are considered to be useful leads for the rational design of new antitumor agents.

Molecular mechanism of action of 2-ferrocenyl-1,1-diphenylbut-1-ene on HL-60 leukemia cells.

The aim of this work was to investigate the mechanism of action of 2-ferrocenyl-1,1-diphenylbut-1-ene (1) on HL-60 human leukemia cells. While inactive against noncancerous cells, 1 provoked a concentration-dependent decrease in viable tumor cells, primarily via apoptosis, as evidenced by analysis of cell morphology, activation of caspases 3 and 7, increased DNA fragmentation, and externalization of phosphatidylserine. Necrosis was observed only at the highest tested concentration (4 µM). Compound 1 interfered with the cell cycle, causing an accumulation of cells in the G1 /G0 phase. Interaction of 1 with dsDNA and ssDNA was observed by differential pulse voltammetry and confirmed by hyperchromicity in the UV/Vis spectra of dsDNA, with an interaction constant of 2×10(4) M(-1). Both the organic analogue 1,1,2-triphenylbut-1-ene (2) and ferrocene were inactive against cancer and noncancer cell lines and did not react with DNA. These results reinforce the idea that the hybrid strategy of conjugating ferrocene to the structure of tamoxifen derivatives is advantageous in finding new substances with antineoplastic activity.

Antifungal activity of naphthoquinoidal compounds in vitro against fluconazole-resistant strains of different Candida species: a special emphasis on mechanisms of action on Candida tropicalis.

In recent decades, the incidence of candidemia in tertiary hospitals worldwide has substantially increased. These infections are a major cause of morbidity and mortality; in addition, they prolong hospital stays and raise the costs associated with treatment. Studies have reported a significant increase in infections by non-albicans Candida species, especially C. tropicalis. The number of antifungal drugs on the market is small in comparison to the number of antibacterial agents available. The limited number of treatment options, coupled with the increasing frequency of cross-resistance, makes it necessary to develop new therapeutic strategies. The objective of this study was to evaluate and compare the antifungal activities of three semisynthetic naphthofuranquinone molecules against fluconazole-resistant Candida spp. strains. These results allowed to us to evaluate the antifungal effects of three naphthofuranquinones on fluconazole-resistant C. tropicalis. The toxicity of these compounds was manifested as increased intracellular ROS, which resulted in membrane damage and changes in cell size/granularity, mitochondrial membrane depolarization, and DNA damage (including oxidation and strand breakage). In conclusion, the tested naphthofuranquinones (compounds 1-3) exhibited in vitro cytotoxicity against fluconazole-resistant Candida spp. strains.

Genetic toxicology evaluation of essential oil of Alpinia zerumbet and its chemoprotective effects against H(2)O(2)-induced DNA damage in cultured human leukocytes.

Essential oil (EO) of Alpinia zerumbet leaves, at non-toxic concentrations (50-300 µg/mL), did not induce genotoxicity in human leukocytes. However, at the highest concentration (500 µg/mL) tested caused a reduction in cell proliferation and viability, and an increase in DNA damage. Moreover, in vivo experiments showed that EO (400 mg/kg) did not exert mutagenicity on peripheral blood cells and bone marrow in mice. In DPPH test, EO showed scavenging effects against DPPH radicals, and other free radicals (determination of intracellular GSH and lipid peroxidation assays). Furthermore, EO was able to reduce the intracellular levels of ROS, and prevented leukocytes DNA against oxidative damage. The ability of EO to reduce H(2)O(2) toxicity was observed only when cells were treated with EO during and after exposure to H(2)O(2). With the co- and post-treatment procedures, EO decreased the frequency of apoptotic and micronucleated leukocytes as well DNA strand breaks. However, a synergistic effect was observed in cultures exposed to 500 µg/mL EO. In conclusion, EO at concentrations up to 300 µg/mL or doses up to 400mg/kg are not mutagenic in leukocytes and in mice, but do have antioxidative and protective effects against the cytotoxicity and clastogenesis induced by H(2)O(2).

Biological evaluation of twenty-eight ferrocenyl tetrasubstituted olefins: cancer cell growth inhibition, ROS production and hemolytic activity.

The antiproliferative effects of twenty-eight tetrasubstituted olefins bearing a ferrocenyl group, including six never-reported compounds, were evaluated against SF-295 (human glioblastoma), HCT-8 (human colon cancer), MDA-MB-435 (human melanoma) and HL-60 (human promyelocytic leukemia) using the MTT test. IC(50) values were determined for twenty-three active compounds and of these, ten compounds had IC(50) values lower than 2 µM on one or more cell lines. Of all the compounds, only two produced significant amounts of ROS on HL-60 cells, and ROS production and growth inhibition could not be correlated. The ten most antiproliferative compounds were tested for their hemolytic activity on mouse erythrocytes. Five compounds showing high antiproliferative activity and low hemolytic activity were thus identified for further study.

Cytotoxic profile of natural and some modified bufadienolides from toad Rhinella schneideri parotoid gland secretion.

Cutaneous secretions of toad species are an important source of bufadienolides, compounds that exhibit interesting structural features and biopharmacological properties. Here we describe the isolation of bufadienolides from the Brazilian toad Rhinella schneideri parotoid glands secretion, including: marinobufagin (1), bufalin (2), telocinobufagin (3), hellebrigenin (4), and the atypical 20S,21R-epoxymarinobufagin (5) besides the widespread beta-sitosterol (6). Starting from natural bufadienolides four derivatives were prepared: 3beta-acetoxy-marinobufagin (7), 3beta-acetoxy-bufalin (8), 3beta-acetoxy-telocinobufagin (9), and 3beta-acetoxy-20S,21R-epoxymarinobufagin (10). The cytotoxic evaluation showed that all natural bufadienolides and their derivatives exhibited moderate to strong activity against human HL-60, SF-295, MDA-MB-435, and HCT-8 cancer cell strains without hemolysis of mouse erythrocytes. The acetylated bufadienolides (7-9) and the epoxide 10 showed lesser peripheral blood lymphocytes (PBLs) inhibitory activity than their precursors, suggesting that chemical modifications on such compounds can play an important role on the modulation of their cytotoxic profile.