A comparative study on the interaction of the putative anticancer alkaloids,sanguinarine and chelerythrine,with single- and double-stranded,and heat-denatured DNAs

A detailed investigation on the interaction of two benzophenanthridine alkaloids, sanguinarine (SGR) and chelerythrine (CHL), with the double-stranded (ds), heat-denatured (hd), and single-stranded (ss) DNA was performed by spectroscopy and calorimetry techniques. Binding to the three DNA conformations leads to quenching of fluorescence of SGR and enhancement in the fluorescence of CHL. The binding was cooperative for both of the alkaloids with all the three DNA conformations. The binding constant values of both alkaloids with the ds DNA were in the order of 10⁶ M^?1; binding was weak with hd and much weaker to the ss DNA. The fluorescence emission of the alkaloid molecules bound to the ds and hd DNAs was quenched much less compared to those bound to the ss DNA based on competition with the anionic quencher KI. For both double stranded and heat denatured structures the emission of the bound alkaloid molecules was polarized significantly and strong energy transfer from the DNA bases to the alkaloid molecules occurred. Intercalation of SGR and CHL to ds, hd, and ss DNA was proved from these fluorescence results. Calorimetric studies suggested that the binding to all DNA conformations was both enthalpy and entropy favored. Both the alkaloids preferred double-helical regions for binding, but SGR was a stronger binder than CHL to all the three DNA structures.     

Immune Response of Mice to Orally Administered Lactic Acid Bacteria

In order to elucidate the interaction of lactic acid bacteria with the immune system, immune responses to the lactic acid bacteria, Bifidobacterium longum and Lactobacillus acidophilus, were examined in mice fed with each organism. In mice fed with B. longum for more than 8 weeks, an antibody response was detected to the cytoplasm of B. longum, but not to the cell wall. On the other hand, in mice fed with L. acidophilus for more than 6 weeks, an antibody response was detected to both the cytoplasm and cell wall of L. acidophilus. Moreover, feeding each organism for 2 weeks enhanced the proliferative response of Peyer’s patch (PP) cells to the cell fraction against which the serum antibody was detected. However, this was not found with spleen cells. These results suggest that mucosal stimulation by lactic acid bacteria may induce a systemic immune response to them.     

Spatial and temporal distribution of the alkaloid sanguinarine in Sanguinaria canadensis L. (Bloodroot)

Acclimated and non-acclimated potted plants of Sanguinaria canadensis L. were harvested at early and late dormancy, anthesis, and immature and mature fruiting stages. Sanguinarine content and concentration were determined for rhizomes (distal, proximal, and middle sections), roots, leaves, flower, and fruit. Rhizomes had highest sanguinarine content and concentrations, and exhibited decreasing concentration gradients from the distal to proximal third. Concentrations in roots were a tenth of rhizome concentration. Concentrations in leaves, flowers, and fruit were one-thousandth of rhizome Sanguinarine content in whole acclimated plants was constant. Content in whole nonacclimated plants increased as the plant became physiologically active, but was constant during fruit maturation: content in roots, leaves, and fruit did not change. The substantial increase in whole-plant dry weight coupled with the unchanging sanguinarine content during fruit maturation suggests either a shift in photosynthate allocation from defense to growth, or a constant turnover of sanguinarine.     

Fluorescence quenching investigation on the interaction of glutathione‐CdTe/CdS quantum dots with sanguinarine and its analytical application

Water‐soluble glutathione (GSH)‐capped core/shell CdTe/CdS quantum dots (QDs) were synthesized. In pH 5.4 sodium phosphate buffer medium, the interaction between GSH‐CdTe/CdS QDs and sanguinarine (SA) was investigated by spectroscopic methods, including fluorescence spectroscopy and ultraviolet‐visible absorption spectroscopy. Addition of SA to GSH‐CdTe/CdS QDs results in fluorescence quenching of GSH‐CdTe/CdS QDs. Quenching intensity was in proportion to the concentration of SA in a certain range. Investigation of the quenching mechanism, proved that the fluorescence quenching of GSH‐CdTe/CdS QDs by SA is a result of electron transfer. Based on the quenching of the fluorescence of GSH‐CdTe/CdS QDs by SA, a novel, simple, rapid and specific method for SA determination was proposed. The detection limit for SA was 3.4 ng/mL and the quantitative determination range was 0.2–40.0 µg/mL with a correlation coefficient of 0.9988. The method has been applied to the determination of SA in synthetic samples and fresh urine samples of healthy human with satisfactory results. Copyright © 2013 John Wiley & Sons, Ltd.     

In situ extraction strategy affects benzophenanthridine alkaloid production fluxes in suspension cultures of Eschscholtzia californica

The effect of contact between cells and extractive phase on secondary metabolite production was investigated in two-phase suspension cultures of Eschscholtzia californica. A system was designed to extract benzophenanthridine alkaloids from the cell culture, without contact between XAD-7 resins and the cells: only medium was recirculated through a column packed with the extractive phase. This strategy was compared to the classic method of addition of resins directly into the cell suspension. Removal of the product directly from the medium enabled important increases in production of alkaloids, namely a 20-fold increase in sanguinarine production and a 10-fold increase in chelerythrine, with high recovery in the resin. The recirculation strategy greatly simplified the production process since the resins are easily recovered from the cell culture and enable harvest of product without termination of culture. However, due to limited flow rate, the recirculation strategy was slightly less effective than direct addition of resins into the cell suspension. In addition to enabling increased production, removal of secondary metabolites from the medium changed metabolic flux distribution, testifying to a complex control mechanism of production.     

The effect of chelerythrine on cell growth, apoptosis, and cell cycle in human normal and cancer cells in comparison with sanguinarine

We compared the effects of chelerythrine (CHE) and sanguinarine (SA) on human prostate cancer cell lines (LNCaP and DU-145) and primary culture of human gingival fibroblasts. CHE and SA treatment of cell lines for 24 h resulted in (1) inhibition of cell viability in a dose-dependent manner in all tested cells (as evaluated by MTT test and bromodeoxyuridine incorporation assay); (2) dose-dependent increase in DNA damage in all tested cells (as evaluated by DNA comet assay); (3) changes in apoptosis (assessed by western blot analysis and TUNEL assay); and (4) significant induction of cyclin kinase inhibitors p21^Waf1/Cip1 and p27^Kip1 in prostate cancer cells (identified by western blot analysis). Our study demonstrates that CHE had significant cytotoxic effect, independent of p53 and androgen status, on human prostate cancer cell lines. Normal gingival fibroblasts and DU-145 cells were more sensitive to the treatment with both alkaloids than were LNCaP cells. CHE and SA may be prospective natural molecules for use in the treatment of prostate cancer owing to their involvement in apoptosis and cell cycle regulation.     

Chelerythrine and sanguinarine dock at distinct sites on BclXL that are not the classic BH3 binding cleft

The ratio of the levels of pro-survival and pro-apoptotic members of the Bcl-2 protein family is thought to be an important regulatory factor for determining the sensitivity of the mammalian cells to apoptotic stimuli. High levels of expression of pro-survival members such as Bcl(XL) in human cancers were frequently found to be a good prognostic indicator predicting poor response to chemotherapy. The pro-survival members of the Bcl-2 family mediate their effects through heterodimerization with the BH3 region of the pro-apoptotic members. Structural analyses of the binding complex of the BH3 peptide and Bcl(XL) showed that a hydrophobic groove termed the BH3 binding cleft is the docking site for the BH3 region. Chemical mimetics of the BH3 region such as BH3I-1 that target the BH3 binding cleft indeed exhibit pro-apoptotic activities. Chelerythrine (CHE) and sanguinarine (SAN) are natural benzophenanthridine alkaloids that are structurally homologous to each other. CHE was previously identified as an inhibitor of Bcl(XL) function from a high-throughput screen of natural products, but its mode of interaction with Bcl(XL) is not known. By determining the effect of site-directed mutagenesis on ligand binding and using saturation transfer difference (STD) NMR experiments, we have verified locations of these docked ligands. Surprisingly, CHE and SAN bind separately at the BH groove and BH1 region of Bcl(XL) respectively, different from the BH3 binding cleft where other known inhibitors of Bcl(XL) target. Interestingly, certain residues on the flexible loop between helices alpha1 and alpha2 of Bcl(XL) are also perturbed upon CHE, but not SAN or BH3I-1 binding. Although CHE and SAN are similarly effective as BH3I-1 in displacing bound BH3 peptide, they are much more effective in inducing apoptosis, raising the possibility that CHE and SAN might be able to antagonize other pro-survival mechanisms in addition to the one that involves BH3 region binding.     

Effect of polymeric adsorbents on the production of sanguinarine by Papaver somniferum cell cultures

The suitability of adsorbent polymeric resins, Amberlite XAD-4 and XAD-7 (Rohm and Hass, Inc.), was investigated for the accumulation of sanguinarine from Papaver somniferum cell cultures. The adsorption and desorption of sanguinarine from aqueous solution was most effective with XAD-7. In addition to sanguinarine, the resins were found to absorb growth regulators and vitamins from the culture medium. Growth inhibition was overcome by delaying for approximately 4 days resin addition after cell inoculation in fresh medium. Resin addition (5% wt/vol) to actively growing uneclicited cultures led to increases in sanguinarine production and release of 30% to 40% and 60%, respectively. The addition of resins to elicited cultures led to increases in alkaloid production of up to 50% to 85% with similar increases in alkaloid release as observed for nonelicited cells. Overall yield of sanguinarine increased from 21 mg . g biomass dry weight(-1) (dw) for elicited cultures to more than 39 mg . gdw(-1) when elicitation was combined with resin addition. Higher quantities of resin (10% to 20% wt/vol) increased marginally the release of sanguinarine into the medium, and on the resin, up to 85% of total production. The use of resin appears promesing for the development of a bioprocess for sanguinarine production by cultured plant cells. (c) 1992 John Wiley & Sons, Inc.     

Seed Dormancy in Acer: An assessment of the Rôle of the Structures Covering the Embryo

Cells of a seven year old strain of Papaver somniferum L. when cultured for 2 weeks and incubated with substances known to elicit the formation of phytoalexins, responded by turning reddish brown within 6 h and accumulating sanguinarine. Morphinan alkaloids were not detected. Media (100 ml) containing 1 ml of Botrytis spec. preparations raised the level of sanguinarine in the cells 26 times over the maximum level found in controls. Over a culture period of 79 h the cells achieved a sanguinarine concentration of 2.9% of dry weight. Media (100 ml) with 1 ml of Rhodotorula rubra preparation, 15 mg arachidonic acid, 1 mg actinomycin, 0.5 ml of Helminthosporium gramineum, Sclerotinia sclerotiorum, or 5 ml Colletotrichum gloeosporoides preparation elicited a considerable, but relatively weaker response. Sanguinarine accumulation was also found to occur in the medium and reached a concentration of 43% of total sanguinarine per culture when cells were cultured in 100 ml medium with 5 ml Colletotrichum preparation for 24 h. Young poppy cell cultures initiated 9 months ago responded to the presence of Botrytis material as did 7-year-old cultures.     

Molecular basis of recognition of quadruplexes human telomere and c-myc promoter by the putative anticancer agent sanguinarine

Background

Interaction of putative anticancer agent sanguinarine with two quadruplex forming sequences, human telomeric DNA (H24) and NHE III₁ upstream of the P1 promoter of c-myc (Pu27), has been studied to understand the structural basis of the recognition.

Methods

Absorption, fluorescence and circular dichroism spectroscopy have been employed to characterize the association. Energetics of the interaction was studied by isothermal titration and differential scanning calorimetry. TRAP assay was done to assess the inhibitory potential of sanguinarine.

Results

Absorption and fluorescence studies show that sanguinarine has high binding affinity of ~ 10⁵ M^− 1 for both sequences. Binding stoichiometry is 2:1 for H24 and 3:1 for Pu27. Results suggest stacking interaction between planar sanguinarine moiety and G-quartets. Circular dichroism spectra show that sanguinarine does not cause structural perturbation in the all-parallel Pu27 but causes a structural transition from mixed hybrid to basket form at higher sanguinarine concentration in case of H24. The interaction is characterized by total enthalpy–entropy compensation and high heat capacity values. Differential scanning calorimetry studies suggest that sanguinarine binding increases the melting temperature and also the total enthalpy of transition of both quadruplexes. TRAP results show that sanguinarine effectively blocks telomerase activity in a concentration dependent manner in cell extracts from MDAMB-231 breast cancer cell lines.

Conclusion

These results suggest that there is a difference in the structural modes of association of sanguinarine to the quadruplexes.

General significance

It helps to understand the role of quadruplex structures as a target of small molecule inhibitors of telomerase.