Oxidation of an antitumor drug ellipticine by peroxidases

Ellipticine is a potent antineoplastic agent, whose mode of action is considered to be based mainly on DNA intercalation and/or inhibition of topoisomerase II. Since we found that ellipticine also forms the cytochrome P450 (CYP)-mediated covalent DNA adducts, this anticancer drug is considered to function as a pro-drug, whose pharmacological efficiency and/or genotoxic side effects are dependent on its enzymatic activation in target tissues. Here, we demonstrate that ellipticine is also oxidized by peroxidases, which are abundantly expressed in several target tumor tissues. Lactoperoxidase, myeloperoxidase and horseradish peroxidase were used as models. Peroxidases in the presence of hydrogen peroxide oxidize ellipticine to an ellipticine dimer and N(2)-oxide of ellipticine as the major and minor metabolite, respectively. Inhibition of the peroxidase-mediated ellipticine oxidation by radical scavengers ascorbate, glutathione and NADH suggests a one-electron mechanism of the oxidation. The implication of the oxidation of ellipticine by peroxidases in its mechanism of action is discussed.     

Interaction between SV40 DNA and camptothecin, an antitumor alkaloid

Camptothecin specifically interacted with closed superhelical circular SV40 DNA during incubation in 1.0 M NaCl at 37 degrees C and induced an alkali-labile linkage in the E strand. No interaction occurred in the reaction mixture containing 0.1 M NaCl, or at 4 degrees C. As camptothecin did not affect linear SV40 DNA, the superhelical structure of DNA appeared to be essential. The site of the alkali-labile linkage induced in SV40 DNA I through interaction with camptothecin was near the origin of replication on the basis of the results of experiments with restriction enzymes. Neither sulfhydryl reagents nor EDTA affected the interaction between camptothecin and SV40 DNA I, so the action of camptothecin is different from those of antitumor antibiotics, bleomycin or neocarzinostatin. Analysis of the s20,0w value of SV40 DNA I after the interaction with camptothecin and the sedimentation profiles of DNA after heating in the reaction mixture indicated that the interaction between camptothecin and SV40 DNA I was different from those of intercalating or alkylating agents such as ethidium bromide and methylmethanesulfonate. Replacement of the OH group at C-20 in the E ring of camptothecin by H-, CH3-, and Cl- resulted in the reduction, in this order, of the potency for interaction with SV40 DNA I to induce an alkali-labile linkage.     

Microspectrofluorometry of carcinogens in living cells

A microspectrofluorometric approach has been used to follow the changes undergone by the carcinogen benzo(a)pyrene in malignant L cells, inducible Buffalo rat liver (BRL) cells and oncogenic mouse embryo C3H/10 T 1/2, clone 8 (CCL 226) cells. Since it is known that benzo(a)pyrene (BP) is converted metabolically to at least 40 metabolites, including phenols, epoxides, quinones, dihydrodiols, diol epoxides, and water-soluble conjugates, the interpretation of blue- and red-spectral shifts in fluorescence emission observed in BP-treated cells, compared to the original BP emission, undoubtedly presents considerable difficulties, but a certain number of facts clearly emerge. The sequence of blue-red shifts expressive of intracellular interactions and detoxification of the carcinogen is accelerated in the induced BRL compared to non-induced, and it is also generally accelerated in the malignant and inducible lines compared to the oncogenic line. The detection of highly reactive molecules (? of ultimate carcinogens) representing a small fraction of bulk fluorescence, still remains elusive, but two promising approaches are described: the use of phase-specific fluorescence quenchers which enable us to probe for the presence of metabolites in aqueous, hydrophobic or membrane phases of the cell, and the matrix analysis based on plotting of excitation-emission at different wavelengths for resolution of complex spectra. The former approach has enabled some separation or enhancement of red-blue emissions, and the second has helped to differentiate between emission of BP per se and its intracellular conversion products. Finally, observations at nuclear and cytoplasmic sites open the possibility of studying carcinogen interactions at different target sites.     

Multimodal action of antitumor agents on DNA: the ellipticine series

Most cytotoxic anticancer agents interact directly or indirectly with nuclear DNA, the ultimate target for this class of compounds. For a given type of drug both direct and indirect action at the DNA level usually causes various types of interference or damage. This multimodal mechanism of action is well illustrated by antitumor drugs in the ellipticine series which may bind to DNA through intercalation, may undergo covalent binding, may generate oxidizing species, and may interfere with the catalytic activity of topoisomerase II. The antitumor activity of these compounds may, therefore, result from alternative cytotoxic events. The present review summarizes information obtained with ellipticine compounds on the relation between the nature of the drugs' action on DNA and their cytotoxic and/or antitumor activity. The occurrence of topoisomerase-mediated DNA cleavage appears to be responsible for antitumor activity. The capability of the drugs to interfere with the action of topoisomerase II requires the presence of an oxidizable phenolic group on their structure. This feature (or a related one) is shared by all antitumor drugs acting on this enzyme.     

Binding of ellipticine to beta-lactoglobulin. A physico-chemical study of the specific interaction of an antitumor drug with a transport protein

The unprotonated form of the anti-tumor alkaloid ellipticine binds to beta-lactoglobulins A and B from bovine milk with an affinity constant of 7 +/- 3 x 10(5) M-1. There is one binding site/dimeric protein molecule (the stable form at medium pH). The attachment site is not the beta-barrel nor the hydrophobic site identified as the retinol site in beta-lactoglobulin but a domain located at the interface of the two monomeric units where the ligand lies close to Trp61 of both polypeptide chains. The positive binding enthalpy observed in temperature-jump relaxation experiments is overcome by a strong entropy increase, tentatively thought to result from water release at the binding domain. Accordingly, desolvation is assumed to be the rate-determining step in the process of ellipticine binding.     

Clastogenic effect of the plant alkaloid ellipticine on bone marrow cells of Wistar rats and on human peripheral blood lymphocytes

Ellipticine (EPC), a natural alkaloid extracted from Aspidosperma williansii (Apocynaceae), is known to have antitumor and cytotoxic activities on various types of tumors. This drug showed a strong clastogenic effect on bone marrow cells of Wistar rats treated in vivo (7.75-31.00 mg/kg body weight). EPC was also tested in vitro using the human peripheral blood lymphocyte system, at concentrations 100 times lower than those used in the in vivo test on rats, since the cytotoxic effect on lymphocytes was very strong. At the 2 highest concentrations used (7.75 X 10(-1) and 1.55 X 10(-1) micrograms/ml culture medium), EPC induced a statistically significant increase in the frequency of chromosome aberrations and sister-chromatid exchanges in lymphocytes. Based on data reported in the literature, we have tried to establish relationships between the clastogenic effect observed and the process of EPC intercalation into DNA and the formation of protein-associated DNA-strand breaks probably promoted by topoisomerase enzymes.     

Microspectrofluorometry of ultraviolet-inducible fluorescence in supravitally-stained ascites tumour cells

Synopsis Ultraviolet irradiation of tumour cells (Ehrlich tetraploid ascites tumour of mice, TO strain), supravitally stained with thiazine dyes (Azure II, Azure A, Methylene Blue, Toluidine Blue) or an oxazine dye (Brilliant Cresyl Blue), induces blue fluorescence in cytoplasmic bodies believed to be lipid droplets or lysosome-like bodies. Microspectrofluorometry of the inducible fluorescence in Ehrlich tumour cells gives bimodal excitation (340/394 nm) and emission (443/700 nm) curves.     

The protonation state of the catalytic aspartates in plasmepsin II

Assigning the correct protonation state to the catalytic residues is essential for a realistic modelling of an enzyme's active site. Plasmepsins are pharmaceutically relevant aspartic proteases involved in haemoglobin degradation by Plasmodium spp. In aspartic proteases, one of the two catalytic aspartates is protonated, while the other is negatively charged. Here, multiple explicit-water molecular dynamics simulations of plasmepsin II, uncomplexed and with a hydroxypropylamine peptidomimetic inhibitor, indicate that protonation of Asp214 favours a stable active site structure. Moreover, the protonation state of the catalytic aspartate has a strong influence on a linear chain of hydrogen bonds with the adjacent side chains.     

Microspectrofluorometry of fluorescent photoproducts in photosensitized cells. Relationship to cellular quiescence and senescence in culture

Microspectrofluorometry of L and WI-38 cells reveals chemical/structural changes due to quiescence or senescence, i.e., lipid peroxidation, spontaneous or photosensitized by hematoporphyrin. Cells treated with hematoporphyrin and a lysosomal umbelliferone probe show a fast-rising umbelliferone emission, plus a fluorescent photoproduct. Studies in rapidly growing versus quiescent L, early passage/late passage WI-38 cells, suggest accumulation of fluorescence Schiff bases (i.e., their association with granular regions of cells in stationary phase, spectral properties, fast increase in photosensitized cells) and a possible lysosomal membrane permeabilization in quiescent or senescent cells.     

Microspectrofluorometry of autofluorescence emission from human leukemic living cells under oxidative stress.

Image cytometry was applied to study the intracellular localization of autofluorescence and the influence of an oxidative stress on this emission. K562 erythroleukemia cancer cells were analyzed with a microspectrofluorometer, coupled with a Argon laser (Ar+) (363 nm). From each cell, 15 x 15 emission spectra were recorded in the 400-600 nm spectral range to generate a spectral image of autofluorescence. The intracellular locations of the autofluorescence emission and of the specific mitochondrial probe rhodamine 123 (R123) were matched. Under a 363 nm excitation, all spectra from K562 cells show equivalent profiles with a 455 nm maximum emission, near of reduced nicotinamide adenine dinucleotide-(Phosphate) solution (NAD(P)H) (465 nm maximum emission). The spatial distribution of autofluorescence is homogeneous and different from the one of R123. Hydrogen peroxide (H2O2) (200 microM) and menadione (Men) (5 microM) induce a weak spectral change and a decrease in autofluorescence intensity, down to 40% of the initial emission. Doxorubicin (Dox) induces a dose-dependent decrease in autofluorescence emission and a release of intracellular free radicals. When cells were pre-treated 1 h with 1 mM glutathione (GSH), Dox induces a lower free radicals release, no significant variation of autofluorescence intensity and a lower growth inhibitory effect. Images cytometry of autofluorescence suggest that the intracellular NAD(P)H would not be restricted to mitochondrial compartments. The release of free radicals was associated with a decrease in autofluorescence intensity, mainly attributed to NAD(P)H oxidation both inside and outside mitochondria.     

Bioassay-guided isolation of an alkaloid with antiangiogenic and antitumor activities from the extract of Fissistigma cavaleriei root

Fissistigma cavaleriei (Levl) Rehd (Annonaceae) is used as a folklore medicine for treatment of inflammation, arthritis, and tuberculosis by Miao people in China. In the present study, the antiangiogenic activity of F. cavaleriei was investigated. The chorioallantoic membrane of the fertilized hen's egg (CAM assay) was used to determine antiangiogenic activity of the plant extract. Compound (1), a compound with antiangiogenic activity, was isolated by bioassay-guided fractionation from F. cavaleriei for the first time. The structure of compound (1) was elucidated on the basis of spectroscopic methods. Colorimetric COX (ovine) inhibitor screening assay was used to determine its inhibitory effect on COX-1 and COX-2. MTT and Sulforhodamine B assays were used to investigate its cytotoxic effects on tumor cell lines. As a result, compound (1) showed a selectively inhibiting effect on COX-2 and could inhibit the growth of tumor cells in vitro. The antitumor activity of compound (1) was further confirmed by the observation that compound (1) administration significantly inhibited the growth of S-180 cells in mice. Moreover, compound (1) was able to enhance the antitumor activity of doxorubicin in the mice bearing with S-180 cells while combined with doxorubicin. In conclusion, compound (1) is a multi-target molecule and further experimental investigations are needed to determine whether it can be used as a lead molecule for tumor treatment.     

Chromophore protonation state controls photoswitching of the fluoroprotein asFP595

Fluorescent proteins have been widely used as genetically encodable fusion tags for biological imaging. Recently, a new class of fluorescent proteins was discovered that can be reversibly light-switched between a fluorescent and a non-fluorescent state. Such proteins can not only provide nanoscale resolution in far-field fluorescence optical microscopy much below the diffraction limit, but also hold promise for other nanotechnological applications, such as optical data storage. To systematically exploit the potential of such photoswitchable proteins and to enable rational improvements to their properties requires a detailed understanding of the molecular switching mechanism, which is currently unknown. Here, we have studied the photoswitching mechanism of the reversibly switchable fluoroprotein asFP595 at the atomic level by multiconfigurational ab initio (CASSCF) calculations and QM/MM excited state molecular dynamics simulations with explicit surface hopping. Our simulations explain measured quantum yields and excited state lifetimes, and also predict the structures of the hitherto unknown intermediates and of the irreversibly fluorescent state. Further, we find that the proton distribution in the active site of the asFP595 controls the photochemical conversion pathways of the chromophore in the protein matrix. Accordingly, changes in the protonation state of the chromophore and some proximal amino acids lead to different photochemical states, which all turn out to be essential for the photoswitching mechanism. These photochemical states are (i) a neutral chromophore, which can trans-cis photoisomerize, (ii) an anionic chromophore, which rapidly undergoes radiationless decay after excitation, and (iii) a putative fluorescent zwitterionic chromophore. The overall stability of the different protonation states is controlled by the isomeric state of the chromophore. We finally propose that radiation-induced decarboxylation of the glutamic acid Glu215 blocks the proton transfer pathways that enable the deactivation of the zwitterionic chromophore and thus leads to irreversible fluorescence. We have identified the tight coupling of trans-cis isomerization and proton transfers in photoswitchable proteins to be essential for their function and propose a detailed underlying mechanism, which provides a comprehensive picture that explains the available experimental data. The structural similarity between asFP595 and other fluoroproteins of interest for imaging suggests that this coupling is a quite general mechanism for photoswitchable proteins. These insights can guide the rational design and optimization of photoswitchable proteins.     

Photosensitization by antitumor agents, 4. Anthrapyrazole-photosensitized formation of single strand-breaks in DNA

Single-strand breaks can be introduced into PM2 closed-circular DNA upon illumination with blue light, in the presence of the anthrapyrazole antitumor agent, compound 1. Damage is observed already after 1 min of blue light illumination, and is significantly enhanced by the presence of electron donors such as NADH, ascorbic acid or Fe(III)/EDTA complex. The photosensitizing properties were not observed for anthrapyrazole analogues with one or more hydroxyl substituents in the chromophore of the drug. The inhibitory effects of sodium azide, methanol, mannitol, SOD, and catalase suggest an oxygen-dependent mechanism of strand-break production, probably involving hydroxyl radicals. However, a second mechanism involving drug molecules bound to the DNA is also indicated under anoxic conditions in the presence of NADH.     

Quantitative identification of the protonation state of histidines in vitro and in vivo

The C[bond]N coupling constants centered at the C(epsilon 1) and C(delta 2) carbons in histidine residues depend on the protonation state and tautomeric form of the imidazole ring, making them excellent indicators of pH or pK(a), and the ratio of the tautomeric states. In this paper, we demonstrate that the intensity ratios for the C(epsilon 1)-H and C(delta 2)-H cross-peaks measured with a constant time HSQC experiment without and with J(C[bond]N) amplitude modulation are determined by the ratios of the protonated and deprotonated forms and tautomeric states. This allows one to investigate the tautomeric state of histidines as well as their pK(a) in situations where changing the pH value by titration is difficult, for example, for in-cell NMR experiments. We apply this technique to the investigation of the bacterial protein NmerA and determine that the intracellular pH in the Escherichia coli cytoplasm is 7.1 +/- 0.1.     

Elaiophylin,a novel autophagy inhibitor,exerts antitumor activity as a single agent in ovarian cancer cells

Currently, targeting the autophagic pathway is regarded as a promising new strategy for cancer drug discovery. Here, we screened the North China Pharmaceutical Group Corporation''s pure compound library of microbial origin using GFP-LC3B-SKOV3 cells and identified elaiophylin as a novel autophagy inhibitor. Elaiophylin promotes autophagosome accumulation but blocks autophagic flux by attenuating lysosomal cathepsin activity, resulting in the accumulation of SQSTM1/p62 in various cell lines. Moreover, elaiophylin destabilizes lysosomes as indicated by LysoTracker Red staining and CTSB/cathepsin B and CTSD/ cathepsin D release from lysosomes into the cytoplasm. Elaiophylin eventually decreases cell viability, especially in combination with cisplatin or under hypoxic conditions. Furthermore, administration of a lower dose (2 mg/kg) of elaiophylin as a single agent achieves a significant antitumor effect without toxicity in an orthotopic ovarian cancer model with metastasis; however, high doses (8 mg/kg) of elaiophylin lead to dysfunction of Paneth cells, which resembles the intestinal phenotype of ATG16L1-deficient mice. Together, these results provide a safe therapeutic window for potential clinical applications of this compound. Our results demonstrate, for the first time, that elaiophylin is a novel autophagy inhibitor, with significant antitumor efficacy as a single agent or in combination in human ovarian cancer cells, establishing the potential treatment of ovarian cancer by this compound.     

Fumschleicherine,an alkaloid of Fumaria schleicheri

The structure of fumschleicherine has been elucidated by spectroscopic methods and confirmed by its conversion to fumaramine.     

Three-layer ONIOM studies of the dark state of rhodopsin: the protonation state of Glu181

A computational three-layer ONIOM(QM-high:QM-low:MM) hybrid scheme has been applied to analyze the protonation state of the Glu181 amino acid residue in rhodopsin, which is vital to determining the rhodopsin photoactivation mechanism. Due to conflicting evidence from previous studies, it has yet to be conclusively resolved. In this study, we fully optimize dark-state rhodopsin model structures differing only at the 181-residue site—protonated and unprotonated Glu181—and calculate several experimentally observable properties. Comparison of calculated structures, excitation energies, and NMR chemical shifts for the two models with values from the literature allows a reevaluation of previously reported conclusions. A key finding is that the S₁ → S₂ energy level splitting, previously used as evidence for a neutral Glu181, is found to be almost identical for the two protonation states. We highlight a need for caution when interpreting experimental data. Small differences in the properties of the two model structures are also identified, which may be useful targets for future high-resolution experimental approaches.     

Distinguishing two groups of flavin reductases by analyzing the protonation state of an active site carboxylic acid

Flavin-containing reductases are involved in a wide variety of physiological reactions such as photosynthesis, nitric oxide synthesis, and detoxification of foreign compounds, including therapeutic drugs. Ferredoxin-NADP(H)-reductase (FNR) is the prototypical enzyme of this family. The fold of this protein is highly conserved and occurs as one domain of several multidomain enzymes such as the members of the diflavin reductase family. The enzymes of this family have emerged as fusion of a FNR and a flavodoxin. Although the active sites of these enzymes are very similar, different enzymes function in opposite directions, that is, some reduce oxidized nicotinamide adenine dinucleotide phosphate (NADP(+)) and some oxidize reduced nicotinamide adenine dinucleotide phosphate (NADPH). In this work, we analyze the protonation behavior of titratable residues of these enzymes through electrostatic calculations. We find that a highly conserved carboxylic acid in the active site shows a different titration behavior in different flavin reductases. This residue is deprotonated in flavin reductases present in plastids, but protonated in bacterial counterparts and in diflavin reductases. The protonation state of the carboxylic acid may also influence substrate binding. The physiological substrate for plastidic enzymes is NADP(+), but it is NADPH for the other mentioned reductases. In this article, we discuss the relevance of the environment of this residue for its protonation and its importance in catalysis. Our results allow to reinterpret and explain experimental data.