Ivana Aljančić

Xanthones from Swertia punctata

Isolation of 1-O-primeverosyl-3,8-dihydroxy-5-methoxyxanthone and 1-O-gentiobiosyl-3,7-dimethoxy-8-hydroxyxanthone, along with five known xanthones, isobellidifolin, methylbellidifolin, isoswertianin, methylswertianin and norswertianin-1-O-beta-D-glucoside, from the roots of Swertia punctata is reported. In the aerial parts four xanthones, bellidifolin, methylbellidifolin, swertianolin and mangiferin, and flavone-C-glucoside, isoorientin were identified. The chemotaxonomic and pharmacological significance of these results is discussed.

Isolation and Biological Evaluation of Jatrophane Diterpenoids from Euphorbia dendroides

From the Montenegrin spurge Euphorbia dendroides, seven new diterpenoids [jatrophanes (1-6) and a tigliane (7)] were isolated and their structures elucidated by spectroscopic techniques. The biological activity of the new compounds was studied against four human cancer cell lines. The most effective jatrophane-type compound (2) and its structurally closely related derivative (1) were evaluated for their interactions with paclitaxel and doxorubicin using a multi-drug-resistant cancer cell line. Both compounds exerted a strong reversal potential resulting from inhibition of P-glycoprotein transport.

Neuropharmacological evaluation of diethylether extract and xanthones of Gentiana kochiana

Diethylether extract of aerial parts of Gentiana kochiana mostly consists of two tetraoxygenated xanthones: gentiacaulein (1,7-dihidroxy-3,8-dimethoxyxanthone; 76.1%) and gentiakochianin (1,7,8-trihidroxy-3-methoxyxanthone; 14.2%). The extract and these xanthones were evaluated for the CNS pharmacological activity in rodents. In vitro assays on rat brain preparations revealed insignificant interaction of the compounds with the specific dopamine and serotonin receptors or synaptosomal uptake of serotonin. However, the extract and gentiacaulein strongly inhibited rat microsomal MAO A (IC50=0.22 microg/ml and 0.49 microM, respectively). Their effects on MAO B and a gentiakochianin blocking potential on both MAO enzymes were moderate. Behavioral examinations on mice showed that 10 day s.c. administration of the extract (20 mg/kg) significantly decreased immobility score in a forced swimming test and strongly inhibited ambulation and stereotypy in an open-field test. These effects resembled those induced by 10 mg/kg imipramine. The ex vivo MAO A activity in crude brain mitochondrial fraction of mice treated with 20 mg/kg of the extract was significantly elevated, whilst that outside brain nerve terminals was declined. This study suggests some antidepressant therapeutic potential of G. kochiana, particularly of gentiacaulein, with an ambiguity whether pharmacological mechanism could be related only to the central inhibition of MAO A.

New anti-cancer characteristics of jatrophane diterpenes from Euphorbia dendroides.

Jatrophane diterpenes were shown to be inhibitors of P-glycoprotein (P-gp). There are also evidences on their microtubule-interacting activity in cancer cells. We evaluated new anti-cancer characteristics of two jatrophane type compounds from Euphorbia dendroides. For that purpose, the model system of sensitive non-small cell lung cancer cell line (NCI-H460) and its resistant counterpart (NCI-H460/R) was used. Although both jatrophanes showed inhibitory effect on cancer cell growth, they were non-toxic for peripheral blood mononuclear cells (PBMC). We examined their effects in combination with paclitaxel (PTX), a well-known mitotic spindle interacting chemotherapeutic. Jatrophanes overcome PTX resistance in concentration-dependent manner in MDR cancer cell line (NCI-H460/R). We observed that this synergistic effect is not caused merely by P-gp inhibition. In combination with PTX, jatrophanes induce cell killing and change cell cycle distribution leading to G2/M arrest. Furthermore, they exert an anti-angiogenic effect by decreasing the vascular endothelial growth factor (VEGF) secretion. The reduction of the level of mdr1 mRNA expression in sensitive cells, suggests that these compounds could not contribute to the development of resistance. In conclusion, present study provides a rational basis for the new cancer treatment approach with jatrophanes that are non-toxic to normal cells and have new favorable anti-cancer characteristics.

In vitro antitumor actions of extracts from endemic plant Helichrysum zivojinii

BackgroundThe aim of this research was to determine the intensity and mechanisms of the cytotoxic actions of five extracts isolated from the endemic plant species Helichrysum zivojinii Černjavski & Soška (family Asteraceae) against specific cancer cell lines. In order to evaluate the sensitivity of normal immunocompetent cells implicated in the antitumor immune response, the cytotoxicity of extracts was also tested against healthy peripheral blood mononuclear cells (PBMC).MethodsThe aerial parts of the plants were air-dried, powdered, and successively extracted with solvents of increasing polarity to obtain hexane, dichloromethane, ethyl-acetate, n-butanol and methanol extracts. The cytotoxic activities of the extracts against human cervix adenocarcinoma HeLa, human melanoma Fem-x, human myelogenous leukemia K562, human breast adenocarcinoma MDA-MB-361 cells and PBMC were evaluated by the MTT test. The mode of HeLa cell death was investigated by morphological analysis. Changes in the cell cycle of HeLa cells treated with the extracts were analyzed by flow cytometry. The apoptotic mechanisms induced by the tested extracts were determined using specific caspase inhibitors.ResultsThe investigated Helichrysum zivojinii extracts exerted selective dose-dependent cytotoxic actions against selected cancer cell lines and healthy immunocompetent PBMC stimulated to proliferate, while the cytotoxic actions exerted on unstimulated PBMC were less pronounced. The tested extracts exhibited considerably stronger cytotoxic activities towards HeLa, Fem-x and K562 cells in comparison to resting and stimulated PBMC. It is worth noting that the cytotoxicity of the extracts was weaker against unstimulated PBMC in comparison to stimulated PBMC. Furthermore, each of the five extracts induced apoptosis in HeLa cells, through the activation of both intrinsic and extrinsic signaling pathways.ConclusionExtracts obtained from the endemic plant Helichrysum zivojinii may represent an important source of novel potential antitumor agents due to their pronounced and selective cytotoxic actions towards malignant cells.

Jatrophane diterpenoids from the latex of Euphorbia dendroides and their anti-P-glycoprotein activity in human multi-drug resistant cancer cell lines

Thirteen jatrophane diterpenoids (1-10, 13-15), three previously isolated (11, 12, 16) and a known tigliane (17) were isolated from the latex of Euphorbia dendroides. The structures and relative configurations of compounds were elucidated by spectroscopic techniques. The P-glycoprotein (P-gp) inhibiting activities of the representative set of jatrophanes (1-6 and 11-16) have been assessed. Jatrophanes 2 and 5 demonstrated the most powerful inhibition of P-gp, higher than R(+)-verapamil and tariquidar in colorectal multi-drug resistant (MDR) cells (DLD1-TxR).

Diterpenes from Achillea clypeolata

The isolation of 16α,17-epoxy-ent-kaurane (a known compound), 3α-acetoxy-16α,17-epoxy-ent-kaurane and 19-acetoxy-16α,17-epoxy-ent-kaurane from roots of Achillea clypeolata is reported.

Molecular and cytogenetic changes in multi-drug resistant cancer cells and their influence on new compounds testing

PurposeMulti-drug resistance (MDR) is a major obstacle to successful cancer treatment. Therefore, in vitro models are necessary for the investigation of the phenotypic changes provoked by cytotoxic agents and more importantly for preclinical testing of new anticancer drugs.MethodsWe analyzed chromosomal, numerical, and structural changes after development of MDR, alterations in p53 and PTEN, single nucleotide polymorphisms (SNPs) in the mdr1 gene and corresponding protein expression of P-glycoprotein (P-gp) in three human MDR cancer cell lines: non-small cell lung carcinoma NCI-H460/R, colorectal carcinoma DLD1-TxR, and glioma U87-TxR. In addition, we explored how these molecular and phenotypic alterations influence the anticancer effect of new drugs.ResultsCytogenetic analysis showed polyploidy reduction after development of MDR in U87-TxR. Losses of 6q in all resistant cancer cell lines and inactivation of p53 in U87-TxR and PTEN in DLD1-TxR were also revealed. Overexpression of P-gp was observed in all MDR cancer cell lines. We evaluated the anticancer activities and MDR reversal potential of Akt inhibitor GSK690693, Ras inhibitor Tipifarnib, and two P-gp inhibitors (jatrophane diterpenoids). Their effects vary due to the cell-type differences, existence of MDR phenotype, presence of mdr1 SNP, and tumor suppressors’ alterations. Tipifarnib and jatrophane diterpenoids significantly sensitized MDR cancer cells to paclitaxel.ConclusionIn conclusion, investigated MDR cancer cells obtained new molecular and cytogenetic characteristics that may serve as potential clinical prognostic markers. In addition, these MDR cancer cell lines present a valuable model for preclinical evaluation of new anticancer agents.

Radioprotective Effects of Gentianella austriaca Fractions and Polyphenolic Constituents in Human Lymphocytes

The aim of this study was to identify active principles of Gentianella austriaca responsible for the reduction of the incidence of micronuclei in irradiated lymphocytes in vitro. The radioprotective effects of ether (EF) and methanolic (MeF) fractions, water-soluble xanthones demethylbellidifolin (1), demethylbellidifolin 8-O-glucoside (2), bellidifolin 8-O-glucoside (3), and flavonoid swertisin (4) against chromosomal damage induced by gamma-rays were determined using the micronucleus test. EF and MeF showed better protection in treatment of human lymphocytes after gamma-irradiation than did isolated compounds. Among the isolated compounds, the effectiveness in reduction of the frequency of micronuclei followed the order 4>3>2>1. The anti-lipoperoxidant activity was in the order 2>4>1, while 3 slightly increased the level of malondialdehyde. In addition, the effectiveness in induction of apoptosis followed the order, 3>2>4, while 1 had no proapoptotic effect. These results suggest that the antioxidative properties of the polyphenols tested may contribute to the radioprotective effects of G. austriaca.

Two structurally distinct chalcone dimers from Helichrysum zivojinii and their activities in cancer cell lines

Dimers tomoroside A (1) and tomoroside B (2) of the co-occuring known chalcone monomer (3), along with the seven known flavonoid glucosides (4-10), were isolated from the aerial parts of Helichrysum zivojinii Černjavski & Soška. The structures of the isolated compounds were elucidated by spectroscopic techniques. Compound 1 inhibited topo IIα and hif-1α expression and stimulated doxorubicin anticancer effect, while 2 increased the expression of hif-1α, probably acting as antioxidant and redox status modulator. Notably, 2 synergized with Tipifarnib showing potential to improve the action of this new chemotherapeutic involved in the modulation of mitogene activated protein (MAP) kinase signaling pathway.