Eva Táborská

Interaction of benzo[c]phenanthridine and protoberberine alkaloids with animal and yeast cells

We compared the effects of four quaternary benzo[c]phenanthridine alkaloids – chelerythrine, chelilutine, sanguinarine, and sanguilutine – and two quaternary protoberberine alkaloids – berberine and coptisine – on the human cell line HeLa (cervix carcinoma cells) and the yeastsSaccharomyces cerevisiae andSchizosaccharomyces japonicus var. versatilis. The ability of alkaloids to display primary fluorescence, allowed us to record their dynamics and localization in cells. Cytotoxic, anti-microtubular, and anti-actin effects in living cells were studied. In the yeasts, neither microtubules nor cell growth was seriously affected even at the alkaloid concentration of 100 μg/ml. The HeLa cells, however, responded to the toxic effect of alkaloids at concentrations ranging from 1 to 50 μg/ml. IC50 values for individual alkaloids were: sanguinarine IC50 = 0.8 μg/ml, sanguilutine IC50 = 8.3 μg/ml, chelerythrine IC50 = 6.2 μg/ml, chelilutine IC50 = 5.2 μg/ml, coptisine IC50 = 2.6 μg/ml and berberine IC50 >10.0 μg/ml. In living cells, sanguinarine produced a decrease in microtubule numbers, particularly at the cell periphery, at a concentration of 0.1 μg/ml. The other alkaloids showed a similar effect but at higher concentrations (5–50 μg/ml). The strongest effects of sanguinarine were explained as a consequence of its easy penetration through the cell membrane owing to nonpolar pseudobase formation and to a high degree of molecular planarity.

Antitumour activities of sanguinarine and related alkaloids

Sanguinarine is a best-known member of a relatively small group of quaternary benzo[c]phenanthridine alkaloids (QBAs). QBAs are widely distributed in the family Papaveraceae and, to a limited extent, in some species of the families Fumariaceae and Rutaceae. From a medical perspective, QBAs have many important properties. In addition to antitumour activity, they display antimicrobial, antifungal and anti-inflammatory effects. They may interact with many targets, such as DNA and microtubules, and they modify the activities of a wide variety of enzymes. This review summarises the current state of knowledge about the properties of QBAs that are important for their potential use in anticancer therapy.

New and facile method of preparation of the anti-HIV-1 agent,1,3-dicaffeoylquinic acid

A facile and inexpensive preparation of 1,3-dicaffeoylquinic acid (cynarin) from the leaves Cynara cardunculus L. (Asteraceae) without the use any chromatographic steps is described. The procedure was based on separation of the fraction rich in 1,5-dicaffeoylquinic acid, isomerisation of 1,5-dicaffeoylquinic acid to cynarin and, owing to its higher polarity, the simple isolation of cynarin from the reaction mixture. Cynarin inhibited HIV-1 replication in MT-2 cell culture at nontoxic concentrations similar to other previously tested dicaffeoylquinic acids, which have been recently established as a potent and highly selective class of HIV-1 integrase inhibitors.

Benzo[c]phenanthridine alkaloids exhibit strong anti-proliferative activity in malignant melanoma cells regardless of their p53 status

BACKGROUND Search for new substances with antiproliferative activity towards melanoma cells is important since malignant melanoma is notoriously resistant to conventional chemotherapy. Benzo[c]phenanthridine alkaloids (BAs) are natural products with significant anti-proliferative activities, therefore they are considered as agents promising for cancer therapy. OBJECTIVES The effects of five BAs (sanguinarine, chelerythrine, chelidonine, sanguilutine, and chelilutine) on human malignant melanoma cell lines were compared. The study focused on BAs effects on DNA, anti-apoptotic and p53 protein levels; and the involvement of p53 in cellular responses to alkaloids treatment. METHODS Melanoma cell lines, two wild types and two with dysfunctional p53 derived from one of them were used. The mechanism of anti-proliferative and pro-apoptotic effects and the effect on DNA was investigated using MTT assay, flow cytometry, Western blot analysis, fluorescence and electron microscopy. RESULTS All tested alkaloids exhibit strong anti-proliferative activity. CHL, CHE and SA induced apoptosis, which was probably mediated by decreasing levels of anti-apoptotic proteins (Bcl-xL, Mcl-1, XIAP) and was accompanied by mitochondrial membrane potential decrease as well as caspase-3 and PARP cleavage. Although all alkaloids caused DNA damage, which was demonstrated by induction of H2AX phosphorylation, none of the tested alkaloids stabilised p53 and their toxicity in cells with non-functional p53 was comparable to wild type cells. CONCLUSION Despite the profound similarity of BAs molecular structures, it is clear that the mechanism of cell death induction is different for each alkaloid. Our results indicate that BAs could be effective in malignant melanoma treatment, including tumours which have lost wild type p53.

Alkaloids and organic acids content of eight Fumaria species

The isoquinoline alkaloids adlumiceine, adlumidiceine, coptisine, corytuberine, cryptopine, fumaricine, fumariline, fumaritine, fumarophycine, O-methylfumarophycine, palmatine, parfumine, protopine, sinactine, stylopine, and N-methylstylopine were determined by reversed phase high performance liquid chromatography in Fumaria agraria, F. capreolata, F. densiflora, F. muralis, F. officinalis, F. parviflora, and F. vaillantii. Organic acids, namely citric, coumaric, ferulic, fumaric, malic, 3-hydroxybenzoic, protocatechuic acid and caffeic acid (and its methylester) were identified by gas chromatography—mass spectrometry. The total content of phenolic constituents was quantified by a colorimetric method using a phosphomolybdic–phosphotungstic reagent. The chemotaxonomic significance of the results is discussed. Copyright

Fluorescence properties of selected benzo[c]phenantridine alkaloids and studies of their interaction with CT DNA

The spectral, especially fluorescence properties, of seven selected quaternary benzo[c]phenantridine alkaloids (sanguinarine, chelerythrine, chelirubine, sanguirubine, chelilutine, sanguilutine, and macarpine) were studied in presence and in absence of double-stranded DNA. This study has proved dramatic differences in fluorescence emission of all studied alkaloids in presence of calf thymus DNA in comparison to fluorescence of free alkaloids. The most remarkable are changes in emission spectra of macarpine, chelirubine, and sanguirubine. Association constants (logK) for interaction of all studied alkaloids with CT DNA were calculated.

Effect of quaternary benzo[c]phenanthridine alkaloids sanguilutine and chelilutine on normal and cancer cells

Sanguilutine and chelilutine, quaternary benzo[c]phenanthridine alkaloids, were studied for their antiproliferative activities with regard to their ability to induce oxidative stress. We observed potent antiproliferative activities for both alkaloids against three tumour (HeLa; HL-60; A-2780) and two normal (V-79; LEP) cell lines. Both alkaloids were efficient inductors of apoptosis. Statistical analysis revealed higher toxicity for sanguilutine compared to chelilutine and unequal sensitivity with regard to individual cell lines, although independent of the character of the cell line (i.e. tumour vs. normal). Dihydrofluorescein diacetate staining was used to measure intracellular ROS accumulation after treatment with sanguilutine, chelilutine, sanguinarine, and chelerythrine. In addition, anti-oxidative effects were studied. The effects of the alkaloids were compared with the effects of commonly used anti-oxidants, such as trolox, caffeine acid, and chlorogenic acid. None of the tested alkaloids (0.1 and 1 microg/ml) increased ROS production. Pre-incubation of sanguinarine and chelilutine (at all tested concentrations) and sanguilutine and chelerythrine (1 microg/ml) decreased oxidative stress caused by H(2)O(2). These findings indicate high antiproliferative and pro-apoptotic effects of sanguilutine and chelilutine that are not accompanied by oxidative stress induction, to the contrary, both alkaloids showed anti-oxidative effects.

Quaternary benzo[c]phenanthridine alkaloids--novel cell permeant and red fluorescing DNA probes.

Quaternary benzo[c]phenanthridine alkaloids (QBAs) are naturally occurring compounds isolated from plants in the Fumariaceae, Papaveraceae, Ranunculaceae, and Rutaceae families. In addition to having a wide range of biological activities, they are also attractive for their fluorescent properties. We observed interesting fluorescent characteristics in the QBAs–macarpine (MA), sanguirubine (SR), chelirubine (CHR), sanguilutine (SL), chelilutine (CHL), sanguinarine (SA) and chelerythrine (CHE) after interaction with living cells.

Screening of Minor Benzo(c)phenanthridine Alkaloids for Antiproliferative and Apoptotic Activities

Abstract Quaternary benzo(c.)phenanthridine alkaloids are a relatively small group of isoquinoline alkaloids with attractive biological activities. They are produced by a number of plant species of the Papaveraceae, Fumariaceae, and Rutaceae families. Differential cytotoxicity of minor, naturally occurring derivatives sanguirubine, chelirubine, and macarpine and better known benzophenanthridines sanguinarine and chelerythrine in cancer and normal cells was assessed in vitro. by MTT assay using a panel consisting of either transformed cell lines (HeLa; A431; HL60) or primary fibroblasts of human origin. IC50 values were determined 72 h after addition of the alkaloids. A wide range (0.01–1.44 µg/mL) of IC50 was observed, and the highest toxicity was determined for macarpine. Human promyelocytic leukemia line HL60 has been documented to be the most sensitive to alkaloid treatment followed by human skin fibroblasts, while human cervix adenocarcinoma HeLa cells and epidermal carcinoma cells A431 appeared more resistant. The most sensitive cell line HL60 was selected for demonstrating the ability of the alkaloids to induce apoptosis by analyzing morphologic changes upon DAPI staining, ultrastructural changes, and annexin V versus propidium iodide staining assay. Apoptosis was induced by sanguirubine, chelirubine, chelerythrine, and macarpine at a concentration of 1 µg/mL. The results show that the alkaloids tested have a strong antiproliferative effect in vitro. due predominately to apoptosis.

Seasonal Variation of Bioactive Alkaloid Contents in Macleaya microcarpa (Maxim.) Fedde

Macleaya microcarpa (Maxim.) Fedde belongs to the genus Macleaya, family Papaveraceae. Together with the better known and more frequently studied species M. cordata (Willd.) R. Br. it is a main source of quaternary benzo[c]phenanthridine alkaloids. Using HPLC we determined the content of eight isoquinoline alkaloids in the aerial and underground parts of 1-, 2-, 12- and 13-year old plants and followed their changes during the vegetative period. The dominant alkaloid of all samples collected in the end of this period was allocryptopine (3.8–13.6 mg/g for aerial parts, 24.2–48.9 mg/g for underground parts). Chelerythrine, sanguinarine and protopine were also present in both parts of the plant. Additionally, measurable concentrations of chelilutine (CL), chelirubine (CR), macarpine (MA) and sanguirubine (SR) were detected in underground parts. The most important finding was that contents of CR, CL, SR and MA in the 12- and 13-year old plant roots were significantly higher (approximately 3-fold for CR, 6-fold for CL, 5-fold for SR, and at least 14-fold for MA) than in 1- or 2-year old plants. The proportion of individual alkaloids in aerial and underground parts thus changed significantly during the vegetative period.