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.

Interferon lambda 2 promotes mammary tumor metastasis via angiogenesis extension and stimulation of cancer cell migration.

Myeloid-derived suppressor cells (MDSCs) support tumor development by stimulation of angiogenesis and immune response inhibition. In our previous study, we showed that interferon lambda 2 (IFN-λ2), secreted by MDSCs, enhances production of pro-angiogenic factors by cancer cells via phosphorylation of STAT3 and therefore promotes blood vessels formation. In the present study IFN-λ2 level was evaluated by ELISA in serum of tumor-bearing mice, whereas its expression in MDSCs isolated from the lungs with metastatic tumors and normal lungs was assessed by qPCR. The effect of IFN-λ2 on mouse mammary cancer cells motility was tested in Boyden chamber migration assay. In order to evaluate its pro-angiogenic function we performed in vitro tubule formation assay and in ovo angiogenesis assay on chicken embryo chorioallantoic membrane (CAM). Moreover, in order to design small molecule inhibitors of IFN-λ2 and its receptor we performed molecular modeling followed by the identification of potential natural inhibitors. Then, we examined their ability to inhibit angiogenesis in vitro. Our results showed that IFN-λ2 predisposed mouse mammary cancer cells to migration in vitro. It also enhanced angiogenesis induced by mouse mammary cancer cells in vitro and in ovo. For the first time we selected potential IFN-λ2 inhibitors and we validated that they were capable to abolish pro-angiogenic effect of IFN-λ2, similarly to blocking antibodies. Therefore, IFN-λ2 and its receptor may become targets of anti-cancer therapy, but their mechanism of action requires further investigation.

Niosomal approach to brain delivery: Development, characterization and in vitro toxicological studies.

The majority of active agents do not readily permeate into brain due to the presence of the blood-brain barrier and blood-cerebrospinal fluid barrier. Currently, the most innovative and promising non-invasive strategy in brain delivery is the design and preparation of nanocarriers, which can move through the brain endothelium. Niosomes can perform brain delivery, in fact polysorbates, can act as an anchor for apolipoprotein E from blood plasma. The particles mimic LDL and interact with the LDL receptor leading to the endothelial cells uptake. The efficacy of niosomes for anticancer therapeutic applications was correlated to their physicochemical and drug delivery properties. Dimensions and ζ-potential were characterized using dynamic light scattering and asymmetric flow-field fractionation system. Lipid bilayer was characterized measuring the fluidity, polarity and microviscosity by fluorescent probe spectra evaluation. Morphology and homogeneity were characterized using atomic force microscopy. Physicochemical stability and serum stability (45% v/v fetal bovine and human serum) were evaluated as a function of time using dynamic light scattering. U87-MG human glioblastoma cells were used to evaluate vesicle cytotoxicity and internalisation efficiency. From the obtained data, the systems appear useful to perform a prolonged (modified) release of biological active substances to the central nervous system.

A p-quinol isoflavan and two new isoflavanones from Desmodium canum.

Three further minor isoflavonoid components (1-3) of the dichloromethane extract of Desmodium canum root have been isolated: two of them are complex isoflavanones with structures 1 and 2 biogenetically related to the previously isolated compounds 1a and 2a. The third is a complex isoflavan where the isoprenyl substituent and the aromatic A-ring yielded a p-quinol nucleus.

Purification and characterization of an antifungal thaumatin-like protein from Cassia didymobotrya cell culture.

A 23-kDa antifungal thaumatin-like protein was isolated and purified from Cassia didymobotrya (Fres.) cell cultures for the first time. The protein was secreted in the culture medium, but it could be also isolated after elution of whole cells with a 0.5 M CaCl(2) solution. Treatment of the cells with laminarin oligosaccharides or salicylic acid, but not with NaCl, resulted in enhancement of expression of the protein. A rapid purification protocol was used based on cationic exchange chromatography. The protein, with a highly basic character (pI 10), has an exact molecular mass of 23034 Da, as determined by MALDI-ToF mass spectrometry analysis. N-terminal sequencing of the intact polypeptide and the sequencing of two internal tryptic peptides indicated significant identity with other thaumatin-like proteins (TLP). The protein exerted antifungal activity towards some Candida species showing EC(50) values comparable to those of other antifungal TLPs. The collected data lead to classify this TLP as a new PR-5 protein.

Triterpenoids and ellagic acid derivatives from in vitro cultures of Camptotheca acuminata Decaisne.

The metabolic profile of secondary products in calli and cell suspension cultures of Camptotheca acuminata Decaisne was investigated and compared to that of the leaves and roots taken from the plant. Neither in vitro system produced the anticancer quinoline alkaloid camptothecin (CPT); they accumulated discrete quantities of polyhydroxylated triterpenoids, different from those found in the plant organs, and ellagic acid derivatives. Nine ellagic acid derivatives (1a-1d and 2a-2e) and eight triterpenoid acids (3a-3e and 4a-4c) were isolated and characterised. All the identified triterpenes were related to ursane- or oleanane-type skeletons and their concentrations rose to 4.5% in suspended cells.

Three isoflavanones with cannabinoid-like moieties from Desmodium canum.

Three further derivatives of 5,7,2',4'-tetrahydroxy-6-methyl isoflavanone have been isolated from the root extract of Desmodium canum and assigned the structures 2,3-dihydro-5,7-dihydroxy-6-methyl-3-(1a,2,3,3a,8b,8c-hexahydro-6-hydroxy-1,1,3a-trimethyl-1H-4-oxabenzo[f]cyclobut[c,d]inden-7-yl)-4H-1-benzopyran-4-one (1) 2,3-dihydro-5,7-dihydroxy-6-methyl-3-(6a,7,8,10a-tetrahydro-3-hydroxy-6,6,9-trimethyl-6H-dibenzo[b,d]pyran-2-yl)-4H-1-benzopyran-4-one (2) 2,3-dihydro-5,7-dihydroxy-6-methyl-3-(3-hydroxy-6,6,9-trimethyl-6H-dibenzo[b,d]pyran-2-yl) 4H-1-benzopyran-4-one (3). The three compounds and the previously isolated chromene 4 all derive from the geranylated precursor 5 by a series of cannabinoid-like oxidative rearrangements.

Aryltetralin lignans: chemistry, pharmacology and biotransformations.

Podophyllotoxin derivatives like etoposide 7a, etophos 7b, and teniposide 7c are used clinically as potent chemotherapeutic agents for a variety of tumors including small cell lung carcinoma, testicular cancer, and malignant lymphoma. These compounds derived from a series of modifications which converted podophyllotoxin 1a from an entity that interacted with tubulin and blocks mitosis to one that induced a block in late S or early G2 by interacting with topoisomerase II. Synthetic studies on podophyllotoxin derivatives can be divided in four general approaches (the oxo-ester route, the digydroxy acid route, the tandem conjugate addition route and the Diels-Alder route). Albeit a number of synthetic sequences afforded products with excellent enantiopurities, the low overall yields still disqualify synthesis as an alternative for naturally produced materials. An alternative route based on the enzyme-catalyzed cyclization of synthetic intermediates to analogues of the podophyllotoxin family is being explored. Synthetic dibenzylbutanolides, which were revealed by biosynthetic studies to be the precursors of aryltetralin lignans, have been treated with enzymes derived from cell cultures of Podophyllum peltatum, Catharanthus roseus, Nicotiana sylvestris and Cassia didymobotrya. The ciclyzation process afforded however compounds with a different stereochemistry in the C ring. The obtainment of a novel compound with a bynzylidenebenzylbutirolactone structure still leaves considerable scope for exploring biotransformations in order to obtain podophyllotoxin analogues via a combination of synthetic chemistry and biotechnological methods.

Synthesis and biosynthesis of isocordoin.

In the search of a convenient synthesis for isocordoin (1), a potential anticancer natural product, 2',4'-dihydroxychalcone was inoculated in cell suspension cultures of Morus nigra, which were expected to contain an active prenyltransferase. After 24 hours the target compound was easily isolated from the metabolite extract. Optimization of the biotransformation resulted in a 85% yield of the prenyl derivative.

Novel hypotensive agents from Verbesina caracasana. 8. Synthesis and pharmacology of (3,4-dimethoxycinnamoyl)-N(1)-agmatine and synthetic analogues.

The more polar metabolites from the Venezuelan plant Verbesina caracasana, i.e., N(3)-prenylagmatine, (3,4-dimethoxycinnamoyl)-N(1)-agmatine, agmatine, and galegine (prenylguanidine), previously reported (Delle Monache, G.; et al. BioMed. Chem. Lett. 1999, 9, 3249-3254), have been synthesized following a biosynthetic strategy. The pharmacologic profiles of various synthetic analogues of (3,4-dimethoxycinnamoyl)-N(1)-agmatine (G5) were also analyzed, to shed some light on the structure-activity relationship of these compounds. Derivatives with the (E)-configuration and/or with a p-methoxybenzoyl moiety were found to be responsible for higher hypotensive effects, which were associated with a slight and, in some cases, not dose-related increase of cardiac inotropism, with variable and not significant chronotopic responses, and, only at higher doses, with effects of respiratory depression. Either an increase (to six) or a decrease (to two) of the number of methylene groups in the alkyl chain of (E)-G5 did not change blood pressure responses, while slightly increasing the positive inotropic ones. At pharmacological doses, all the studied compounds showed hypotensive and slight positive inotropic effects without relevant chronotropic and respiratory actions.

Further hypotensive metabolites from Verbesina caracasana.

After the isolation of caracasanamide and caracasandiamide, further hypotensive components of Verbesina caracasana were shown to be N3-prenylagmatine, N1-3',4'-dimethoxycinnamoylagmatine, agmatine and galegin (prenylguanidine). The structures were assigned on the basis of the spectral data of both metabolites and products from their alkaline hydrolyses. A pharmacological analysis of these products is also presented.

Novel hypotensive agents from Verbesina caracasana. 6. Synthesis and pharmacology of caracasandiamide.

Caracasandiamide, a second hypotensive agent isolated from Verbesina caracasana, is the cyclobutane dimer (truxinic type) of the previously reported 1-[(3, 4-dimethoxycinnamoyl)amino]-4-[(3-methyl-2-butenyl)guanidino]butane (caracasanamide) (Delle Monache, G.; et al. BioMed. Chem. Lett. 1992, 25, 415-418). The structure was confirmed by synthesis starting from beta-truxinic acid obtained by photoaddition of 3, 4-dimethoxycinnamic acid. The dimer was coupled with 2 mol of prenylagmatine to give caracasandiamide in satisfactory yield. By contrast, the direct photodimerization of caracasanamide was unsuccessful. Caracasandiamide, assayed by the iv route in anesthetized rats at doses ranging from 50 to 3200 microgram/kg of body weight, was found to have no appreciable effect on heart rate. At lower doses, the drug stimulates breathing and increases cardiac inotropism, stroke volume, and cardiac output, thus augmenting blood pressure and aortic flow. At higher doses, caracasandiamide depresses breathing likely through central neurogenic mechanisms (not involved in the cardiovascular effects), continues to stimulate cardiac inotropism, and induces, by reducing peripheral vascular resistance, arterial hypotension with reduction of both aortic flow and stroke volume. These cardiovascular effects appear to involve complex interactions at the level of the peripheral beta(1)-, beta(2)-, and alpha(2)-adrenoreceptor-dependent as well as M(2)- and M(4)-cholinergic receptor-dependent transductional pathways both in cardiovascular myocells and at the level of the postganglionic sympathetic endings (with reserpine- and guanethidine-like mechanisms). The cardiovascular effects of caracasandiamide, different from those of caracasanamide, do not depend on significant actions on the central nervous system and on baroreflex pathways. In a similar manner and more effective than caracasanamide, caracasandiamide may be considered a hypotensive and antihypertensive drug. It is devoid of some of the negative side effects, e.g., reflex tachycardia and decreased cardiac inotropism, which are shown by the majority of the most common antihypertensive and vasodilator drugs.

Human cystic echinococcosis in a Uruguayan community: a sonographic, serologic, and epidemiologic study.

A prevalence and transmission study of human cystic echinococcosis (CE), due to infection with the dog tapeworm Echinococcus granulosus, was undertaken in the village of La Paloma in central Uruguay. The human population was registered and screened for CE by abdominal ultrasound scan as well as a number of serologic tests. Dogs were screened for E. granulosus infection by arecoline purgation as well as specific coproantigen testing. The total prevalence of human CE (new cases and those with a previous history) was 5.6% (64 of 1,149); 3.6% (40) of the cases were new ultrasound detected asymptomatic cases (mean age = 45 years). Age prevalence increased from 1.1% in the 4-6-year-old group to > 11% in the > 60-year-old group; the 20-29-year-old group had a significantly higher CE rate of 7.4%, compared with younger and older age groups, and there was no difference between sexes. A CE rate of 3.9% (20 of 514) was also recorded by ultrasound for new cases in the population residing outside the village. Most of the hydatid cysts were located in the liver presenting as either univesicular cysts or a solid mass, and of those 71% and 63%, respectively, with such cyst presentations were seropositive against E. granulosus cyst fluid antigens. Two of eight individuals who were filter paper blood spot seropositive, but ultrasound scan negative, were subsequently diagnosed respectively with pulmonary hydatidosis after radiography, and hepatic hydatidosis after computed tomography scan. Of 36 households with a CE patient, 32 were single cases while four households each harbored two CE cases. This did not represent a clustered distribution within families (23 of 117). Almost 20% of the dogs from La Paloma were found infected with E. granulosus after purge examination, with a mean worm number of 67 (range = 1-1,020). An additional eight dogs that were purge negative were Echinococcus coproantigen positive. The study showed that human CE is highly endemic in Uruguay, with one of the highest local prevalence rates in the world. Transmission appears to occur readily within well-developed towns, as well as on rural sheep ranches. Mass screening by ultrasound scanning with confirmatory serologic testing is an effective approach to case detection at the community level.

Glycated human hemoglobin (HbA1c): functional characteristics and molecular modeling studies.

A minor hemoglobin component of human red blood cell hemolysate, HbA1c, is the result of the non-enzymatic reaction of glucose with the alpha-amino groups of the valine residues at the N-terminus of the beta-chains of human hemoglobin. In this paper, the effect of protons, chloride and 2,3-diphosphoglycerate (DPG) on the functional properties of HbA1c has been investigated in some details. Moreover, the structural modifications induced on the native molecule by the sugar moieties, studied by computer modeling, do agree with the observed functional alterations. In particular, the functional results indicate that: (a) the low-affinity conformation (or T-state) of HbA1c is destabilized by the chemical modification per se; (b) the Bohr effect is reduced with respect to that of native HbA0; (c) the affinity of the T-state of HbA1c for 2,3-diphosphoglycerate is about 2.6 x lower than that of the corresponding conformational state of HbA0, while the R-state is less affected with, the affinity being 1.7 x lower. At the structural level, computer modeling studies show that the two sugar moieties are asymmetrically disposed within the 2,3-diphosphoglycerate binding site. In addition, molecular mechanics and dynamics calculations concerning the interaction with 2,3-diphosphoglycerate indicate that while in HbA0 the effector can assume two different stable orientations, in glycated Hb only one orientation is possible. All together, the results show that glycation of the Val 1 residues of both beta-chains does not impair the binding of DPG but imposes a different mode of binding by changing the internal geometry of the complex and the surface distribution of the positive electrostatic potential within the binding pocket.

Purification and partial characterization of a peroxidase from plant cell cultures of Cassia didymobotrya and biotransformation studies.

An acidic peroxidase (EC 1.11.1.7) produced by cell suspension cultures of Cassia didymobotrya (wild senna) was purified from culture medium collected on the 29th day. The enzyme was shown to be a glycoprotein with a pI of 3.5, a molecular mass of approx. 43 kDa by SDS/PAGE and 50 kDa by gel filtration. The N-terminal sequence was very similar to those of other plant peroxidases. The peroxidase was characterized by a high specificity towards coniferyl alcohol and other natural phenolics such as guaiacol and ferulic and caffeic acids. These findings suggest that the enzyme is involved in lignification processes of the cell wall. Moreover, the enzyme was able to catalyse the oxidation of 4,3',4'-trihydroxychalcone and 4, 3',4'-trihydroxy-3-methoxychalcone to the corresponding 3, 3'-biflavanones, as mixtures of racemic and meso forms.

Antimicrobial isoflavanones from Desmodium canum.

Bioassay-directed fractionation of Desmodium canum resulted in the isolation and characterization of three antimicrobial isoflavonones. These compounds, namely, desmodianones A, B and C, were assigned the structures 5,7,2'-trihydroxy-6,6"-dimethyl-6"-(4-methylpent-3- enyl)pyrano(2",3";4',5')isoflavanone, 5,2',4'-trihydroxy-7-methoxy-6-methyl-8-(3-methylbut-2-enyl)-is oflavanone, and 5,7,2',4'-tetrahydroxy-6-methyl-5'-(3,7-dimethylocta-2,6-dienyl )-isoflavanone, respectively.

Histological study of callus formation and optimization of cell growth in Taxus baccata.

Callus growth of Taxus baccata was optimized when Murashige and Skoog or B5 media were used with 2,4-D/kinetin ratios of 1:0.1, 2:0.1, and 5:0.1, when statistically equivalent results were obtained. A cell suspension culture was successfully achieved with a 3-fold increase in fresh weight after 40 days. Histological examination showed that in the leaf explants both the epidermis and mesophyll tissues divided to produce callus. In the stem explants callus formation was due to the cell division of the cortical parenchyma and of the cambium.

Comparison between metabolite productions in cell culture and in whole plant of Maclura pomifera.

Plant tissue cultures of Maclura pomifera showed a metabolite accumulation pattern which was both quantitatively and qualitatively different from that of the parent plant. Triterpenes and flavonoids were isolated from callus and cell cultures, however, xanthones and stilbenes, which have been reported in the whole plant, were not found. Among the flavonoids, flavones and flavanones were produced preferentially by the suspended cells, but with the prenyl substituents exclusively on ring A, while the isoflavones did not show the 3',4'-dihydroxyl substitution pattern found in the products isolated from fruits. A new prenylated 6'-deoxychalcone was also isolated from the callus and cell cultures.

Novel hypotensive agents from Verbesina caracasana. 2. Synthesis and pharmacology of caracasanamide.

Caracasanamide, one of the hypotensive agents isolated from Verbesina caracasana, is a mixture of (Z)-1a and (E)-1b forms of 1-[(3,4-dimethoxycinnamoyl)amino]-4- [(3-methyl-2-butenyl)-guanidino]butane. The structure of (E)-caracasanamide (1b) was confirmed by high-yielding synthesis starting from N,N'-bis(tert-butoxycarbonyl)-S-methylisothiourea. The water-soluble Z-form of 1a, assayed by i.v. route in anesthetized rats at doses ranging from 50 to 1600 micrograms/kg body weight, was found to decrease blood pressure, to increase cardiac inotropism, respiratory frequency, and tidal volume, and to induce a very slight and not significant tachycardia. Higher doses determined respiratory depression and, in some cases, consequent cardiac arrest. The compound was shown to affect cardiovascular function by acting at the vascular level in inducing arterial vasodilation, by determining sympathetic hypotone through central neurogenic mechanisms, and by interacting with the cardiac beta 1-adrenoreceptors. The respiratory effects were independent of the cardiovascular ones. In lowering blood pressure, the compound was more potent than guanethidine and not less potent than reserpine and papaverine. (Z)-Caracasanamide may therefore be useful in the treatment of arterial hypertension of moderate degree.

Inhibition of adenosine 3',5'-cyclic monophosphate phosphodiesterase by flavonoids from licorice roots and 4-arylcoumarins.

Isoliquiritigenin, glabridin, licoarylcoumarin and licoricidin were identified as strong inhibitors of adenosine 3',5'-cyclic monophosphate (cAMP) phosphodiesterase in waste materials which were obtained during the industrial extraction of glycyrrhizin from licorice roots. The structure-activity relationships of 12 flavonoids from licorice roots and 34 4-arylcoumarins were studied. In 4-arylcoumarins, 5,7-dihydroxy derivatives were generally highly inhibitory towards cAMP phosphodiesterase.