Involvement of lipid rafts in the localization and dysfunction effect of the antitumor ether phospholipid edelfosine in mitochondria.

Lipid rafts and mitochondria are promising targets in cancer therapy. The synthetic antitumor alkyl-lysophospholipid analog edelfosine (1-O-octadecyl-2-O-methyl-rac-glycero-3-phosphocholine) has been reported to target lipid rafts. Here, we have found that edelfosine induced loss of mitochondrial membrane potential and apoptosis in human cervical carcinoma HeLa cells, both responses being abrogated by Bcl-x(L) overexpression. We synthesized a number of new fluorescent edelfosine analogs, which preserved the proapoptotic activity of the parent drug, and colocalized with mitochondria in HeLa cells. Edelfosine induced swelling in isolated mitochondria, indicating an increase in mitochondrial membrane permeability. This mitochondrial swelling was independent of reactive oxygen species generation. A structurally related inactive analog was unable to promote mitochondrial swelling, highlighting the importance of edelfosine molecular structure in its effect on mitochondria. Raft disruption inhibited mitochondrial localization of the drug in cells and edelfosine-induced swelling in isolated mitochondria. Edelfosine promoted a redistribution of lipid rafts from the plasma membrane to mitochondria, suggesting a raft-mediated link between plasma membrane and mitochondria. Our data suggest that direct interaction of edelfosine with mitochondria eventually leads to mitochondrial dysfunction and apoptosis. These observations unveil a new framework in cancer chemotherapy that involves a link between lipid rafts and mitochondria in the mechanism of action of an antitumor drug, thus opening new avenues for cancer treatment.

New analogues of the BODIPY dye PM597: photophysical and lasing properties in liquid solutions and in solid polymeric matrices.

New tailormade BODIPY dyes have been synthesized by a simple protocol to reach wavelength finely tunable laser action from 540 to 625 nm while maintaining highly efficient and photostable laser emission. The new dyes are analogues of the commercial dye PM597 with the eight position free (PTH8) or substituted by the groups acetoxymethyl (PTAlk) or p-acetoxymethylphenyl (PTAr). The photophysical properties strongly depend on the geometrical distortion from planarity of the indacene core generated by the presence of the bulky 2,6-di-tert-butyl groups and the eight substituent. In both liquid and polymeric solid solutions, lasing efficiencies of up to 63 and 48%, respectively, were observed under transversal pumping at 532 nm with high photostabilities. In the case of PTAlk incorporated into silicon-containing solid organic matrices, the laser emission remained at 92% of its initial intensity value after 100,000 pumping pulses in the same position of the sample at 30 Hz repetition rate. The laser action of the new dyes enhances that of the parent dye PM597 and outperforms the lasing behavior of dyes considered to be benchmarks over the green-yellow to red spectral region.

Photophysical study of new versatile multichromophoric diads and triads with BODIPY and polyphenylene groups.

The photophysical properties of multichromophoric dyes with borondipyrromethene (BODIPY) and poly- p-phenylene (di- p-phenylene and tri- p-phenylene) groups in the same molecule are studied in detail. The excitation of the polyphenylene moiety in the UV region leads to a strong visible fluorescent emission of the BODIPY chromophore, via intramolecular excitation energy transfer between both groups. Consequently, these multichromophoric dyes are characterized by a large "virtual" Stokes shift, with a high fluorescence capacity and an efficient laser emission. On the other hand, the photophysical properties of a related dichromophoric dye with a hydroxy end group at the di- p-phenylene moiety show an important decrease in the fluorescent emission due to a photoinduced electron transfer process in basic media. Therefore, its photophysical properties are sensitive to the environmental acidity/basicity and could be applied as a proton sensor.

Photophysical and laser emission studies of 8-polyphenylene-substituted BODIPY dyes in liquid solution and in solid polymeric matrices.

In our search for efficient and photostable laser dyes, four new dyes with the basic structure of the commercial BODIPY laser dye PM567, with either an 8-diphenylene or an 8-p-triphenylene group, both substituted at the terminal polyphenylene position with an acetoxymethyl (dyes P2Ar1Ac and P3Ar1Ac, respectively) or a methacryloyloxymethyl group (dyes P2Ar1MA and P3Ar1MA, respectively), have been synthesized. The photophysical and lasing properties of the dyes have been studied both dissolved in liquid solvents (acetoxymethyl dyes) and incorporated into solid polymeric matrices, in the latter case as solutions (acetoxymethyl dyes) or as copolymers with methyl methacrylate (methacryloyloxymethyl dyes). In liquid solution, the photophysics of P2Ar1Ac and P3Ar1Ac is scarcely affected by the number (two or three) of p-phenylene units. Quantum mechanical calculations reveal that the p-phenylene units in these dyes are twisted ca. 37 degrees each other, an that the first 8-p-phenylene group stands nearly perpendicular to the aromatic BODIPY plane, resulting in electronic decoupling of the two chromophores. P2Ar1Ac exhibits a somewhat lower photodegradation quantum yield under UV and visible irradiation, if compared with P3Ar1Ac or with PM567, likely because of its also lower rate constant for the reaction with in situ-generated singlet molecular oxygen. Both acetoxymethyl dyes emit laser radiation in solution in all the solvents tried, under transversal pumping at 532 nm. In ethyl acetate, with a dye concentration of 0.80 x 10(-3) M, laser efficiencies as high as 80% have been observed. When the 8-polyphenylene dyes were incorporated into solid poly(methyl methacrylate) (PMMA) matrices, as solutions or as copolymers, the fluorescence emission increased with respect to that of the parent PM567 dye dissolved in the same matrix, and lasing efficiencies in the range 18-31% were obtained, with good photostability. The dye P2Ar1Ac dissolved in PMMA was found to exhibit the best overall laser behavior, with a good balance between efficiency and photostability.

Photophysical characterization of new 3-amino and 3-acetamido BODIPY dyes with solvent sensitive properties.

The structural, electronic and photophysical properties of three new asymmetric, highly fluorescent difluoroborondipyrromethene (BODIPY) dyes, bearing an amino or an acetamido group at position 3 of the chromophoric core, have been studied in different apolar, polar and polar/protic solvents. The presence of the 3-amido group extents the delocalization of the pi-system, leading to bathochromic shifts in the absorption and fluorescence bands, as predicted by quantum mechanic calculations. The 3-amino dye shows photophysical properties highly dependent on the solvent polarity and acidity, and is characterized by a hypsochromic shift of its absorption band, with regard to the corresponding acetylated dye, as well as a low fluorescence quantum yield in acid media with proton concentration lower than 4 x 10(-4) M. In media with higher proton concentration, the BF(2) bridge group of the 3-amino dye is removed, yielding the corresponding non-fluorescent dipyrromethene precursor. These results suggest that the 3-amino dye could be used as a fluorescence probe for the study of the acidity of different environments.

Photodegradation of the herbicide Norflurazon sensitised by Riboflavin. A kinetic and mechanistic study.

The kinetics and mechanism of the Riboflavin (Rf)-promoted photochemical degradation with visible light of the herbicide Norflurazon (NF) has been studied by time-resolved and stationary techniques. Using light of wavelength higher than 400 nm--a region where NF is totally transparent--and with concentrations of Rf and NF of ca. 0.02 and 1 mM, respectively, only the excited triplet state of Rf ((3)Rf*) is quenched by NF, in competition with dissolved ground state triplet oxygen, O(2)((3)Sigma(g)(-)). NF degradation mainly occurs by reaction with superoxide radical anion O(2)(-) formed through two electron transfer steps: from NF to (3)Rf*, yielding Rf radical anion, and from this anion to O(2)((3)Sigma(g)(-)), regenerating ground state Rf. Although singlet molecular oxygen is also produced, NF only quenches this oxidative species in a physical mode. The global result is the photoprotection of the sensitiser and the photodegradation of NF.

Photophysical and lasing properties of new analogs of the boron-dipyrromethene laser dye pyrromethene 567 incorporated into or covalently bounded to solid matrices of poly(methyl methacrylate).

The photophysical, lasing and thermostability properties of newly synthesized analogs of the commercial dye pyrromethene 567 (PM567) have been measured in polymeric matrices of poly(methyl methacrylate) both when used as a dopant and when covalently bounded to the polymeric chain. These analogs have an acetoxy or a polymerizable methacryloyloxy group at the end of a polymethylene chain at Position 8 of the PM567 chromophore core. Clear correlations between photophysical and lasing characteristics are observed. Linking chain lengths with three or more methylene units give the highest fluorescence quantum yields (as high as 0.89) and lasing efficiencies (as high as 41%). The covalent linkage of the chromophore to the polymeric chain via the methacryloyloxy group improves the photostability of the PM567 chromophore.

Kinetic study of the riboflavin-sensitised photooxygenation of two hydroxyquinolines of biological interest.

4-Hydroxyquinoline (4-OHQ) and 8-hydroxyquinoline (8-OHQ), two compounds of interest because of their bioactivity and their structural relation with bioactive products, are effectively photooxygenated when irradiated with visible light in the presence of riboflavin (Rf) (vitamin B2) in solution in air-saturated water-methanol (9:1). Rf behaves as a dye-sensitiser, since both quinolines are transparent to visible light. 8-OHQ degrades about five times faster than 4-OHQ. Kinetic data obtained through time-resolved and stationary detection of Rf-electronically excited states indicate that a superoxide radical anion-mediated mechanism exclusively operates for 4-OHQ, whereas singlet molecular oxygen--mainly--plus superoxide radical anion is the species that reacts with 8-OHQ. The sensitiser Rf, which is known to photodegrade under visible-light aerobic irradiation, is regenerated in the presence of any of the quinolines through an electron transfer process that produces superoxide radical anion. The overall picture indicates that both quinolines act as sacrificial scavengers of the photogenerated oxygen species, thus preventing the photodegradation of Rf.

Centrosome and spindle pole microtubules are main targets of a fluorescent taxoid inducing cell death.

Microtubules offer a very large local concentration of binding sites for cytotoxic taxoids or for hypothetical endogenous regulators. Several compounds from diverse sources stabilize microtubules and arrest cell division similarly to the antitumour drug Taxol. We have investigated the subcellular location of the Taxol binding sites, employing a fluorescent taxoid (FLUTAX) that reversibly interacts with the Taxol binding sites of microtubules and induces cellular effects similar to Taxol. The specific binding of FLUTAX to a fraction of the available cellular binding sites effectively inhibits division of cultured human tumour cells at G(2)/M, and triggers apoptotic death. The loci of reversible binding, directly imaged in intact U937 cells treated with cytotoxic doses of fluorescent taxoid are the centrosomes, with a few associated microtubules in interphase cells, and the spindle pole microtubules in mitotic cells, instead of uniformly labelling the microtubule cytoskeleton. Cytoskeletal lesions induced and visualized with FLUTAX consist of microtubule bundles and abnormal mitoses, including monopolar spindles and highly fluorescent multipolar spindles. The multiple asters and monopolar spindles mark arrested U937 leukaemia and OVCAR-3 ovarian carcinoma cells on their path to apoptosis (as well as K562, HeLa, and MCF-7 cells). Depending on the FLUTAX treatment, OVCAR-3 cells died from abnormal mitosis or from a subsequent interphase with double chromatin content and lobulated nuclei (micronuclei), indicating impairment of centrosome separation. Fragmented centrosomes could be observed in FLUTAX-treated non-transformed 3T3.A31 cells, which developed micronuclei but were resistant to apoptosis. These results strongly suggest that centrosomal impairment by taxoid binding during interphase, in addition to the suppression of the kinetochore microtubule dynamics in the mitotic spindle, is a primary cause of cell cycle de-regulation and cell death.

Effect of 2'-OH acetylation on the bioactivity and conformation of 7-O-[N-(4'-fluoresceincarbonyl)-L-alanyl]taxol. A NMR-fluorescence microscopy study.

The relationship between conformation, 2'-OH acetylation, and bioactivity of two fluorescent taxoids has been investigated by a combination of NMR and fluorescence microscopy techniques. These taxoids present the structure of taxol with the 7-OH group esterified with the N-(4'-fluoresceincarbonyl)-L-alanine group and with the 2'-OH group free (taxoid 2) or acetylated (taxoid 3). The larger water solubility of 2 and 3 compared with taxol allowed a detailed NMR study in DMSO-d6/D2O (3/7), showing that both taxoids adopt a similar collapsed conformation in which the hydrophobic groups 2-O-benzoyl, 3'-phenyl and 4-O-acetyl are in close proximity, with the fluorescein group displaying unrestricted motion. On the other hand, while taxoid 2 retains essentially the ability of taxol to induce in vitro microtubule assembly and to bind to cell microtubules, the 2'-acetylated derivative 3 does not show immediate activity. However, when taxoid 3 is left in the cell culture, the slow hydrolysis of the 2'-acetate group in the medium liberates the cytotoxic, microtubule-specific taxoid 2. The intense emission of this active derivative (2) allows the accurate recording of the drug-cell interaction from the very initial steps using fluorescence microscopy. These experiments show conclusively, for the first time in cell cultures, that a free 2'-OH group in taxol is essential for the recognition of the drug by the binding site of cellular microtubules.

Fluorescent taxoids as probes of the microtubule cytoskeleton.

Microtubules are specifically and efficiently visualized with the new fluorescent taxoids 7-O-[N-(4'-fluoresceincarbonyl)-L-alanyl]taxol (FLUTAX) and 7-O-[N-(4'-tetramethylrhodaminecarbonyl)-L-alanyl]taxol (ROTAX). Similarly to taxol, FLUTAX and ROTAX are able to drive inactive GDP-liganded tubulin into microtubule assembly. One molecule of FLUTAX binds per alphabeta-tubulin dimer assembled, competing with taxol for the same microtubule binding site with an eightfold smaller relative affinity. FLUTAX-induced microtubule elongation is markedly Mg2+-dependent, encompassing the binding of one Mg2+ ion more per tubulin dimer polymerized than in the case of taxol. A small perturbation of the absorption spectrum of bound FLUTAX is consistent with a cationic microenvironment relative to the solution. The fluorescence anisotropy of FLUTAX increases by an order of magnitude upon binding to microtubules and time-resolved measurements indicate that the fluorescein moiety remains considerably mobile on a protein surface. The rate of labeling suggests that this is the outer microtubule wall. Alternatively, the microtubule lumen would be functional. FLUTAX- and ROTAX-induced microtubules, radial structures, and organized microtubule bundles are readily observed under the fluorescence microscope. Rapid and accurate visualization of native (or very mildly fixed) cytoplasmic and spindle microtubules of a variety of permeabilized cells is simply obtained with micromolar FLUTAX, with an advantage over immunofluorescence. In addition, FLUTAX labels the centrosomes of PtK2 cells more intensely than antibodies to alpha- or beta-tubulin, and co-localizing with antibodies to gamma-tubulin. Two brightly fluorescent spots, probably separating or duplicating centrioles, can be resolved in the centrosomes of interphase cells. This finding indicates that centrosomes may well be additional targets of action of taxoids. FLUTAX strongly labels microtubules near the spindle poles, as well as microtubules at the telophase spindle equator and the central part of the midbody in cytokinesis (instead of the dark zone frequently observed with immunofluorescence), suggesting a predominant interaction of FLUTAX with sites at which tubulin is newly polymerized. Nanomolar concentrations of FLUTAX also permit specific imaging of centrosomes, half-spindles and midbodies in growing U937 cells.

New fluorescent octadecapentaenoic acids as probes of lipid membranes and protein-lipid interactions.

The chemical and spectroscopic properties of the new fluorescent acids all(E)-8, 10, 12, 14, 16-octadecapentaenoic acid (t-COPA) and its (8Z)-isomer (c-COPA) have been characterized in solvents of different polarity, synthetic lipid bilayers, and lipid/protein systems. These compounds are reasonably photostable in solution, present an intense UV absorption band (epsilon(350 nm) approximately 10(5) M(-1) cm(-1)) strongly overlapped by tryptophan fluorescence and their emission, centered at 470 nm, is strongly polarized (r(O) = 0.385 +/- 0.005) and decays with a major component (85%) of lifetime 23 ns and a faster minor one of lifetime 2 ns (D,L-alpha-dimyristoylphosphatidylcholine (DMPC), 15 degrees C). Both COPA isomers incorporate readily into vesicles and membranes (K(p) approximately 10(6)) and align parallel to the lipids. t-COPA distributes homogeneously between gel and fluid lipid domains and the changes in polarization accurately reflect the lipid T(m) values. From the decay of the fluorescence anisotropy in spherical bilayers of DMPC and POPC it is shown that t-COPA also correctly reflects the lipid order parameters, determined by 2H NMR techniques. Resonance energy transfer from tryptophan to the bound pentaenoic acid in serum albumin in solution, and from the tryptophan residues of gramicidin in lipid bilayers also containing the pentaenoic acid, show that this probe is a useful acceptor of protein tryptophan excitation, with R(O) values of 30-34 A.

Proton-transfer lasers from solid polymeric chains with covalently bound 2-(2'-hydroxyphenyl) benzimidazole groups.

The lasing properties of a number of newly synthesized 2-(2'-hydroxyphenyl) benzimidazole derivatives copolymerized with methyl methacrylate or dissolved in poly (methyl methacrylate) and pumped with an N(2) laser are reported. The radiation mechanism is based on the intramolecular proton-transfer in the electronic excited state. Both the lasing efficiency and the dye photostability increase significantly when the proton-transfer chromophore is covalently bound to the polymeric chain, and energy-conversion efficiencies comparable to those obtained in liquid solution are demonstrated.

New photopolymer used as a holographic recording material.

The composition and the holographic behavior of a new photopolymer with high diffraction efficiencies (30% at 514 and 60% at 633 nm) are described.

Application of factor analysis to the study of the forms of succinylfluorescein present in buffer solutions in aqueous methanol.

The pH-dependent forms adopted by the xanthene dye succinylfluorescein in 1:1 v/v aqueous methanol buffers have been deduced from factor analysis of absorbance data measured at six wavelengths in the visible region, and sixteen pH-values. It is concluded that a protonated species and a doubly-charged anion are the only absorbing species at the two ends of the pH-range studied, 1.40-9.77, and the quinonoid neutral and singly charged anion forms, both with similar absorption properties, are the major components in the pH-range 5-6. A colourless minor compound may also be present at around pH 4. Apparent pK values corresponding to the three ionizations involved are 2.90, 4.60 and 6.80, as determined by potentiometry.