Protein-Mediated Carotenoid Delivery Suppresses the Photoinducible Oxidation of Lipofuscin in Retinal Pigment Epithelial Cells.

Lipofuscin of retinal pigment epithelium (RPE) cells is a complex heterogeneous system of chromophores which accumulates as granules during the cell's lifespan. Lipofuscin serves as a source of various cytotoxic effects linked with oxidative stress. Several age-related eye diseases such as macular degeneration of the retina, as well as some severe inherited eye pathologies, are accompanied by a significant increase in lipofuscin granule concentration. The accumulation of carotenoids in the RPE could provide an effective antioxidant protection against lipofuscin cytotoxic manifestations. Given the highly lipophilic nature of carotenoids, their targeted delivery to the vulnerable tissues can potentially be assisted by special proteins. In this study, we demonstrate how protein-mediated delivery of zeaxanthin using water-soluble Bombyx mori carotenoid-binding protein (BmCBP-ZEA) suppresses the photoinducible oxidative stress in RPE cells caused by irradiation of lipofuscin with intense white light. We implemented fluorescence lifetime imaging of the RPE cell culture ARPE-19 fed with lipofuscin granules and then irradiated by white light with and without the addition of BmCBP-ZEA. We demonstrate that after irradiation the mean fluorescence lifetime of lipofuscin significantly increases, while the presence of BmCBP-ZEA at 200 nM concentration suppresses the increase in the average lifetime of lipofuscin fluorescence, indicating an approx. 35% inhibition of the oxidative stress. This phenomenon serves as indirect yet important evidence of the efficiency of the protein-mediated carotenoid delivery into pigment epithelium cells.

Thiophene-2-carboxamide derivatives of anthraquinone: A new potent antitumor chemotype.

The anthraquinone scaffold has long been known as a source of efficacious antitumor drugs. In particular, the various chemical modifications of the side chains in this scaffold have yielded the compounds potent for the wild type tumor cells, their counterparts with molecular determinants of altered drug response, as well as in vivo settings. Further exploring the chemotype of anticancer heteroarene-fused anthraquinones, we herein demonstrate that derivative of anthra[2,3-b]thiophene-2-carboxamide, (compound 8) is highly potent against a panel of human tumor cell lines and their drug resistant variants. Treatment with submicromolar or low micromolar concentrations of 8 for only 30 min was sufficient to trigger lethal damage of K562 chronic myelogenous leukemia cells. Compound 8 (2.5 µM, 3-6 h) induced an apoptotic cell death as determined by concomitant activation of caspases 3 and 9, cleavage of poly(ADP-ribose) polymerase, increase of Annexin V/propidium iodide double stained cells, DNA fragmentation (subG1 fraction) and a decrease of mitochondrial membrane potential. Neither a significant interaction with double stranded DNA nor strong inhibition of the DNA dependent enzyme topoisomerase 1 by 8 were detectable in cell free systems. Laser scanning confocal microscopy revealed that some amount of 8 was detectable in mitochondria as early as 5 min after the addition to the cells; exposure for 1 h caused significant morphological changes and clustering of mitochondria. The bioisosteric analog 2 in which the thiophene ring was replaced with furan was less active although the patterns of cytotoxicity of both derivatives were similar. These results point at the specific role of the sulfur atom in the antitumor properties of carboxamide derivatives of heteroarene-fused anthraquinone.

Amides of pyrrole- and thiophene-fused anthraquinone derivatives: A role of the heterocyclic core in antitumor properties.

Heteroarene-fused anthraquinone derivatives represent a class of perspective anticancer drug candidates capable of targeting multiple vital processes including drug resistance. Taking advantage of previously demonstrated potential of amide derivatives of heteroarene-fused anthraquinones, we herein dissected the role of the heterocyclic core in antitumor properties. A new series of naphtho[2,3-f]indole-3- and anthra[2,3-b]thiophene-3-carboxamides was synthesized via coupling the respective acids with cyclic diamines. New compounds demonstrated a submicromolar antiproliferative potency close to doxorubicin (Dox) against five tumor cell lines of various tissue origin. In contrast to Dox, the new compounds were similarly cytotoxic for HCT116 colon carcinoma cells (wild type p53) and their isogenic p53 knockout counterparts. Modification of the heterocyclic core changed the targeting properties: the best-in-series naphtho[2,3-f]indole-3-carboxamide 8 formed more affine complexes with DNA duplex than furan and thiophene analogs, a property that can be translated into a stronger inhibition of topoisomerase 1 mediated DNA unwinding. At tolerable doses the water soluble derivative 8 significantly inhibited tumor growth (up to 79%) and increased the lifespan (153%) of mice bearing P388 lymphoma transplants. Together with better solubility for parenteral administration and well tolerance by animals of the indole derivative 8 indicates prospects for further search of new antitumor drug candidates among the heteroarene-fused anthraquinones.

Application of Peak Intensity Analysis to Measurements of Protein Binding to Lipid Vesicles and Erythrocytes Using Fluorescence Correlation Spectroscopy: Dependence on Particle Size.

Fluorescence correlation spectroscopy (FCS) is a sensitive analytical tool for investigation of processes accompanied by changes in the mobility of molecules and complexes. In the present work, peak intensity analysis (PIA) in combination with the solution stirring using FCS setup was applied to explore the interaction between fluorescently labeled protein ligands and corresponding receptors located on membranes. In the system composed of biotinylated liposomes and fluorescently labeled streptavidin as a ligand, PIA allowed us to determine the optimum receptor concentration and demonstrate the essential dependence of the binding efficacy on the length of the linker between the biotin group and the polar head group of the lipid. The binding was dependent on the size of liposomes which was varied by lipid extrusion through filters of different pore diameters. The sensitivity of the method was higher with the liposomes of larger sizes. The PIA approach can be applied not only to liposomes but also to relatively large objects, e.g., erythrocytes or Sepharose beads derivatized with lactose as a receptor for the binding of viscumin and ricin.

Photodynamic activity of the boronated chlorin e6 amide in artificial and cellular membranes.

Photodynamic tumor-destroying activity of the boronated chlorin e6 derivative BACE (chlorin e6 13(1)-N-{2-[N-(1-carba-closo-dodecaboran-1-yl)methyl]aminoethyl}amide-15(2), 17(3)-dimethyl ester), previously described in Moisenovich et al. (2010) PLoS ONE 5(9) e12717, was shown here to be enormously higher than that of unsubstituted chlorin e6, being supported by the data on much higher photocytotoxicity of BACE in M-1 sarcoma cell culture. To validate membrane damaging effect as the basis of the enhanced tumoricidal activity, BACE was compared with unsubstituted chlorin e6 in the potency to photosensitize dye leakage from liposomes, transbilayer lipid flip-flop, inactivation of gramicidin A ionic channels in planar lipid membranes and erythrocyte hemolysis. In all the models comprising artificial and cellular membranes, the photodynamic effect of BACE exceeded that of chlorin e6. BACE substantially differed from chlorin e6 in the affinity to liposomes and erythrocytes, as monitored by fluorescence spectroscopy, flow cytometry and centrifugation. The results support the key role of membrane binding in the photodynamic effect of the boronated chlorin e6 amide.

Novel photosensitizers trigger rapid death of malignant human cells and rodent tumor transplants via lipid photodamage and membrane permeabilization.

BACKGROUND: Apoptotic cascades may frequently be impaired in tumor cells; therefore, the approaches to circumvent these obstacles emerge as important therapeutic modalities. METHODOLOGY/PRINCIPAL FINDINGS: Our novel derivatives of chlorin e(6), that is, its amide (compound 2) and boronated amide (compound 5) evoked no dark toxicity and demonstrated a significantly higher photosensitizing efficacy than chlorin e(6) against transplanted aggressive tumors such as B16 melanoma and M-1 sarcoma. Compound 5 showed superior therapeutic potency. Illumination with red light of mammalian tumor cells loaded with 0.1 µM of 5 caused rapid (within the initial minutes) necrosis as determined by propidium iodide staining. The laser confocal microscopy-assisted analysis of cell death revealed the following order of events: prior to illumination, 5 accumulated in Golgi cysternae, endoplasmic reticulum and in some (but not all) lysosomes. In response to light, the reactive oxygen species burst was concomitant with the drop of mitochondrial transmembrane electric potential, the dramatic changes of mitochondrial shape and the loss of integrity of mitochondria and lysosomes. Within 3-4 min post illumination, the plasma membrane became permeable for propidium iodide. Compounds 2 and 5 were one order of magnitude more potent than chlorin e(6) in photodamage of artificial liposomes monitored in a dye release assay. The latter effect depended on the content of non-saturated lipids; in liposomes consisting of saturated lipids no photodamage was detectable. The increased therapeutic efficacy of 5 compared with 2 was attributed to a striking difference in the ability of these photosensitizers to permeate through hydrophobic membrane interior as evidenced by measurements of voltage jump-induced relaxation of transmembrane current on planar lipid bilayers. CONCLUSIONS/SIGNIFICANCE: The multimembrane photodestruction and cell necrosis induced by photoactivation of 2 and 5 are directly associated with membrane permeabilization caused by lipid photodamage.