A High-throughput Screening of a Chemical Compound Library in Ovarian Cancer Stem Cells.

BACKGROUND: Epithelial ovarian cancer has a poor prognosis, mostly due to its late diagnosis and the development of drug resistance after a first platinum-based regimen. The presence of a specific population of "cancer stem cells" could be responsible of the relapse of the tumor and the development of resistance to therapy. For this reason, it would be important to specifically target this subpopulation of tumor cells in order to increase the response to therapy. METHOD: We screened a chemical compound library assembled during the COST CM1106 action to search for compound classes active in targeting ovarian stem cells. We here report the results of the high-throughput screening assay in two ovarian cancer stem cells and the differentiated cells derived from them. RESULTS AND CONCLUSION: Interestingly, there were compounds active only on stem cells, only on differentiated cells, and compounds active on both cell populations. Even if these data need to be validated in ad hoc dose response cytotoxic experiments, the ongoing analysis of the compound structures will open up to mechanistic drug studies to select compounds able to improve the prognosis of ovarian cancer patients.

Evaluation of the repellent effects of Nepeta parnassica extract, essential oil, and its major nepetalactone metabolite against mosquitoes.

The dichloromethane-methanol extract, the essential oil obtained by hydrodistillation from Nepeta parnassica, and the isolated 4aα,7α,7aß-nepetalactone were evaluated for their repellent effect against the mosquitoes Aedes (Stegomyia) cretinus Edwards and Culex pipiens pipiens biotype molestus Forskål. The chemical analysis of N. parnassica essential oil, dominated by oxygenated monoterpenes (87 %), revealed 4aα,7α,7aß-nepetalactone (36.8%), 1,8-cineole (25.5%), and 4aα,7ß,7aß-nepetalactone (11.1%) as the major constituents. The results of the insect bioassays showed that the essential oil and the dichloromethane-methanol extract of N. parnassica were very active against Aedes cretinus for up to 3 h and against Culex pipiens for up to 2 h post application. The isolated 4aα,7α,7aß-nepetalactone showed very high mosquito repellency for periods of at least 2 h against both species.

Inhibition of sensory afferents activation and visceral pain by a brominated algal diterpene.

BACKGROUND: In the search of new therapeutic options for the treatment of pain, isolation, and testing of secondary metabolites from plant extracts has raised significant attention. We have investigated the effects of the brominated diterpene O(11) 15- cyclo-14-bromo-14,15-dihydrorogiol-3,11-diol (that we have named VLC5), extracted from the Mediterranean red algae Laurencia glandulifera. METHODS: The pure extract was tested on primary afferent calcium signals induced by high concentration of KCl, transcient receptor potential vanilloid (TRPV)1 (capsaicin) or TRPV4 agonists, histamine, or protease-activated receptor-2 (PAR(2) ) agonist. It was also tested in mice in a model of mustard oil-induced colonic hypersensitivity. KEY RESULTS: VLC5 was inhibited PAR(2) agonist or histamine-induced calcium mobilization in mouse primary afferents, but did not modify calcium signals induced by high concentrations of KCl, TRPV1 or TRPV4 agonists. The effect of VLC5 on histamine-induced calcium signal in primary afferent was inhibited by pertussis toxin pretreatment and was dependent on the activation of mu- or kappa-opioid receptor agonists, as it was inhibited by selective antagonists of those two receptors, but not by selective antagonist of the delta-opioid receptor. Intraperitoneal treatment of mice with VLC5 (10 mg kg(-1)) significantly reduced visceral pain behaviors induced by the intracolonic administration of mustard oil, in an opioid receptor-dependent manner. CONCLUSIONS & INFERENCES: We have demonstrated significant analgesic properties for the algal metabolite VLC5, which is able to signal directly to primary afferents, through a mechanism dependent on the activation of opioid receptors. This identifies a new natural compound capable of activating peripheral opioidergic systems, exerting analgesic properties.

Antimicrobial activity of pentacyclic triterpenes isolated from Acacia mellifera.

Acacia mellifera has been used widely in traditional African medicines against various diseases. Among the Kipsigis community of Kenya, water extracts from the plant is used for the treatment of skin diseases, coughs and gastrointestinal ailments. The aim of the study was to provide scientific rationale for the use of the plant in traditional medicine through bioassay-guided fractionation of A. mellifera stem bark. Bioactivity testing was done against selected microbes using disc diffusion technique as outlined in Clinical Laboratory Standard Institute (CLSI). Structure elucidation of the isolated compounds was based primarily on 1D and 2D NMR analyses, including HMQC, HMBC, and NOESY correlations. Fractionation yielded three triterpenoids; (20S)-oxolupane-30-al, (20R)-oxolupane-30-al, and betulinic acid. The three compounds were active against Staphylococcus aureus ATCC 25923 and only (20S)-oxolupane-30-al against clinical isolate of Microsporum gypseum. The three compounds had no activity against Escherichia coli ATCC 25922, Enterococcus feacalis, Candida albicans ATCC 90028, Cryptococcus neoformans, Trichophyton mentagrophyte, Candida krusei, Microsporum gypseum, and Sacharomyces cerevisiae. These results explain and support the use of A. mellifera stem barks for the treatment of infectious diseases in traditional Kenya medicine. It also shows that the antimicrobial activity is concentrated in the triterpenoid fractions.

In Vivo Screening of Antimalarial Activity of Acacia Mellifera (Benth) (Leguminosae) on Plasmodium Berghei in Mice

The activities of total extracts and lupane triterpenes; isolated from the stem bark of Acacia mellifera;were evaluated against Plasmodium berghei strain ANKA in a female Swiss mouse model. Five isolated compounds and the crude extracts were evaluated for antimalarial activity and Quinine hydrochloride was used as a positive control. Only betulin and the methanolic extract produced considerable antimalarial activity in mice infected with P. berghei parasites. This study demonstrated the presence of bioactive agents in Acacia mellifera

Effect of four genes (ALDH1, NRF1, JAM and KBL) on proliferation arrest in a non-small cell bronchopulmonary cancer line.

Despite new protocols, non-small cell bronchopulmonary cancers are still difficult to treat by current chemotherapeutic procedures. Thus, it is essential to define new treatment strategies and detect new therapeutic targets. In order to define these new targets, this study applied the "differential display" (DD) technique to the NSCLC-N6 cell line treated with VT1 [methyl-4-methoxy-3-(3-methyl-2-butanoyl)benzoate]. VT1 induces arrest of the NSCLC-N6 cell cycle in the G1-phase, followed by cell death. DD enabled us to detect seven overexpressed mRNAs during treatment, four of which corresponded to identified genes: aldehyde dehydrogenase 1, nuclear transcription factor Nrfl, junctional adhesion molecule, and amino-ketobutyrate ligase. An antisense strategy showed that amino-ketobutyrate ligase is involved in the proliferation arrest of NSCLC-N6 cells in the G1-phase after VT1 treatment.

Metallothionein levels in the bivalves Callista chione and Venus verrucosa from two Mediterranean sites.

Metallothioneins levels (MTs) in the clams Callista chione and Venus verrucosa, collected from two coastal sites in Greece, were determined and quantified by SDS polyacrylamide gel electrophoresis (SDS-PAGE) and a spectrophotometric assay (Ellman's reaction). SDS-PAGE separation in the digestive gland, which represents the hepato-pancreas in clams, demonstrated the presence of MTs similar to mammalian MT (rabbit liver Cd, Zn-thionein). No other SH-containing proteins apart from the MTs were detected. MT levels quantified by the Ellman's reaction indicated seasonal variation for both species. The highest values were recorded in the spring and the lowest in the autumn. The seasonal variation and the differences in the MT levels of the two areas seem to be related to the reproductive cycle of the organisms as well as to abiotic factors of each area. Our results show that both C. chione and V. verrucosa have the potential to be used as biomarkers of metal pollution, provided that the influence of the external factors is safely quantified.

Foliar and cortex oleoresin variability of silver fir (Abies alba Mill.) in Albania.

Terpene composition of needle and cortical oleoresin from lateral shoots were analyzed by GC/MS for four Silver fir (Abies alba Mill.) populations scattered in natural species range in Albania. More than sixty compounds were detected in the needle oleoresin, which was characterized by a high content of alpha-pinene, camphene, beta-pinene, limonene and bornyl acetate. Three monoterpenes, alpha-pinene, beta-pinene and limonene, and two sesquiterpenes, beta-caryophyllene and germacrene D, comprised the majority of cortical oleoresin. The terpene composition differences among the populations that led to the recognition of two chemotypes. The needle oleoresin from the provinces of Puka, Bulqiza and Llogara were characterised by high amounts of beta-pinene, camphene and alpha-pinene and low amounts of limonene, while that from Drenova had high amounts of beta-pinene and limonene. A similar pattern was found in the cortical oleoresin with the exception of camphene that was a minor contributor. Geographical and seasonal variation between the populations was, also, investigated. Multivariate analysis of both needle and cortical oleoresin separated Drenova (southeastern population) from the other sites. When both major monoterpenes and sesquiterpenes were considered four chemical profiles could be attributed. Based on their chemical profiles, the populations can be divided into two groups: Populations with high content of beta-pinene and alpha-pinene but a low content of limonene (Puka, Bulqiza and Llogara), typical of most of A. alba populations in all its distribution range. Population with a high content of limonene and a moderate content of beta-pinene and alpha-pinene (Drenova).

Chemical composition and behavioral responses of the marine insect Halobates hawaiiensis (Heteroptera: Gerridae).

Halobates is the only insect genus with representatives in the open ocean. How adults find one another at sea has long been an intriguing issue. Since chemical communications have been demonstrated in a related marine veliid Trochopus, and laboratory bioassays indicated behavioral differences between males and females when insect extracts were presented, we carried out similar studies on Halobates. Analyses of surface lipid constituents of female and male Halobates hawaiiensis revealed marked differences. Palmitic and oleic acid, major constituents in the male extracts, were absent in the female extract, whereas nonacosenol, dominating the female extracts, was not detected in the male extracts. Analyses of nymphal extracts indicated an intermediate chemical profile. Surface waxes of all insect stages investigated showed nonacosanol and isononacosanol to be main components. "Headspace" analyses of airborne chemicals showed high levels of 4-hydroxy-4-methyl-2-pentanone and benzaldehyde from the male, whereas benzyl alcohol was the main component in the female mixture.

Chemical intra-Mediterranean variation and insecticidal activity of Crithmum maritimum.

The chemical composition of the volatile metabolites of Crithmum maritimum harvested from several geographic localities along the Mediterranean coasts was studied by GC and GC-MSD. The major oil constituents were found to be dillapiole, gamma-terpinene, sabinene, limonene and beta-phellandrene. The Western populations were richer in dillapiole, whereas the Southern collections were characterized by increased amounts of thymol methyl ether and gamma-terpinene. The Italian chemical profiles differentiated by the significant contributions of carvacrol methyl ether and isoterpinolene. The essential oils were also investigated for their insecticidal activity and their repellency against Pheidole pallidula (Nylander) ants and found to possess significant activity.

Chemical variability of the volatile metabolites from the Caribbean corals of the genus Gorgonia.

The chemical composition of the investigated gorgonians showed a high level of individual variation and the colonies, according to their major contributors, were assigned to 10 distinct chemical profiles, among which A, C, E, and G were the most abundant ones. From the metabolites identified in the present study, either by means of GC/MS or using NMR techniques after conventional separation procedures, the novel cyclic ether 5,10-epoxymuurolane is found in significant quantities in D and I chemical profiles. Furanotriene, isofuranotriene and furanodiene could be referred as the most common metabolites of the genus, since they are found in 6 out of 10 chemical profiles. Isosericenine is, also, a significant contributor of H and I chemical profiles. A number of sesquiterpene hydrocarbons, such as curzerene, bicyclogermacrene, valencene, beta-bourbonene and beta-elemene, along with the oxygenated sesquiterpenes elemanolide and furoventalene, are present at varying concentrations in the majority of the chemical profiles. Metabolites of high discriminant value are: alpha-himachalene for the K chemical profile, alpha-santalene and its oxygenated derivatives for the G chemical profile and the three geometrical isomers of germacrone for the F chemical profile. Several chemical profiles showed narrow geographic distribution. Most of the chemical profiles are located in the north, while F inhabits mainly southern sites and the others are equally distributed. Finally, 91% of the chemical profiles of the gorgonian colonies appeared to grow in all depths, while 9% did not inhabit deep-water environments. Most chemical profiles are less frequent at higher water depths with the exception of chemical profiles A and C.

New prostaglandins from the chemically defended soft coral Plexaura nina.

Three new prostaglandins were isolated as minor constituents of the organic extract from the Caribbean soft coral Plexaura nina. The structures of the new natural products were established by means of spectral data analysis, including 2D NMR experiments. The unpalatability of the lipid extract of the coral and the defensive role of the major prostaglandin metabolites were determined by laboratory and field fish-feeding assays.

Antibacterial and cytotoxic natural and synthesized hydroquinones from sponge Ircinia spinosula.

In order to check the structure-activity relationship and find more potent derivatives of the natural products 1 and 2 obtained from sponge Ircinia spinosula, a series of oxidation, hydrogenation, acetylation and methylation derivatives was prepared. All compounds (natural and synthetic ones) were screened for their cytotoxic and antibacterial activities. The biological studies showed a wide range of antibacterial activity even though only 2 and 2d showed a moderate cytotoxicity against the clone C98. The oxidation of the hydroquinone to quinone and the hydrogenation of the side-chain increased the antibacterial effect of the molecules.

Composition and antibacterial activity of the essential oils of Helichrysum rupestre and H. ambiguum growing in the Balearic Islands (Part III)

The chemical compositions of the essential oils obtained from the aerial parts of Helichrysum rupestre and H. ambiguum were analysed by GC and GC/MS. From the forty-five identified constituents representing 90.21% and 93.90% of the two oils, respectively, manoyl oxide, dodecanal, nonanal, p-cymene, alpha-pinene, and 2,4,6-tris(1,1-dimethylethyl)benzoic acid, were found to be the major components. Furthermore, the oils were tested against six Gram (+/-) bacteria and it was found that one of them exhibited significant antibacterial activity.

Antiphage properties of some Greek algae extracts.

The ethanol extracts of 35 algae collected in several Greek shores, were screened for antiphage properties against 6 bacteriophages: coliphages T 1, T 2, T 4, T 7, ΦX 174 and MS 2. Five samples were found to induce a mild antiphage activity.

Chemical and antibacterial studies of two Helichrysum species of Greek origin.

The chemical composition of the essential oils obtained from the aerial parts of Helichrysum stoechas ssp. barrelieri and H. taenari was analysed by GC and GC/MS. From the thirty-nine identified constituents representing the 73.87% and 87.41% of the two oils, respectively, beta-elemene, beta-caryophyllene, geraniol, and camphene were the major components. Furthermore, it was found that the oils exhibited significant antibacterial activity against six Gram (+/-) bacteria.

Chemical and biological studies on two Helichrysum species of Greek origin.

The chemical composition of the essential oils obtained from the aerial parts of Helichrysum amorginum and H.italicum were analysed with GC and GC/MS. From the twenty-five identified constituents representing the 89.98% and 82.06% of the two oils respectively, geraniol, geranyl acetate, neryl acetate, and nerolidol were the major components. Furthermore, it was found that the oils exhibited definite antibacterial activity against the six tested Gram (+/-) bacteria.