Photosensitized co-generation of nitric oxide and singlet oxygen Enhanced toxicity against ovarian cancer cells.

Near micromolar concentrations of nitric oxide (NO) induce tumor cells death. However, an appropriate NO load has to be delivered selectively to the tumor site in order to avoid NO loss and secondary NO-induced effects. The encapsulation of millimolar concentrations of a NO source and an appropriate trigger of NO release within phospatidylcholine-based liposomes should provide an efficient tool for the selective release of the needed NO payload. In this work we report the photosensitized generation of singlet oxygen and NO from folate-targeted PEGylated liposomes, containing AlPcS4 as the sensitizer and S-nitrosoglutathione (GSNO), in millimolar amounts, as the NO source. Amounts of singlet oxygen detected outside the liposome when using PEGylated liposomes are near 200 % larger when GSNO is present inside the liposomes as compared to its absence. These liposomes, conjugated to folate, were found to enhance the photosensitized cytotoxicity to A2780CP20 ovarian cancer cells as compared to liposomes containing the sensitizer but no GSNO (30 % as compared to 70 % cell viability) under the conditions of this work. Fluorescense of AlPcS4 was observed inside cells incubated with folate-conjugated liposomes but not with liposomes without folate. The photosensitized activity enhancement by GSNO increased when light fluence or liposome concentration were increased. The majority of ovarian cancer patients are initially diagnosed with disseminated intra-abdominal disease (stages III-IV) and have a 5-year survival of less than 20%. This work suggests a novel ovarian cancer nodules treatment via the use of tumor-targeted liposome nanoparticles with the capability of generating simultaneously reactive oxygen and nitrogen species upon illumination with near-infrared light.

Synthesis and Evaluation of Folate-Conjugated Phenanthraquinones for Tumor-Targeted Oxidative Chemotherapy.

Almost all cells are easily killed by exposure to potent oxidants. Indeed, major pathogen defense mechanisms in both animal and plant kingdoms involve production of an oxidative burst, where host defense cells show an invading pathogen with reactive oxygen species (ROS). Although cancer cells can be similarly killed by ROS, development of oxidant-producing chemotherapies has been limited by their inherent nonspecificity and potential toxicity to healthy cells. In this paper, we describe the targeting of an ROS-generating molecule selectively to tumor cells using folate as the tumor-targeting ligand. For this purpose, we exploit the ability of 9,10-phenanthraquinone (PHQ) to enhance the continuous generation of H2O2 in the presence of ascorbic acid to establish a constitutive source of ROS within the tumor mass. We report here that incubation of folate receptor-expressing KB cells in culture with folate-PHQ plus ascorbate results in the death of the cancer cells with an IC50 of ~10 nM (folate-PHQ). We also demonstrate that a cleavable spacer linking folate to PHQ is significantly inferior to a noncleavable spacer, in contrast to most other folate-targeted therapeutic agents. Unfortunately, no evidence for folate-PHQ mediated tumor regression in murine tumor models is obtained, suggesting that unanticipated impediments to generation of cytotoxic quantities of ROS in vivo are encountered. Possible mechanisms and potential solutions to these unanticipated results are offered.

Metal-independent reduction of hydrogen peroxide by semiquinones.

The quinones 1,4-naphthoquinone (NQ), tetramethyl-1,4-benzoquinone (DQ), 2-methyl-1,4-naphthoquinone (MNQ), 2,3-dimethoxy-5-methyl-1,4-benzoquinone (UBQ-0), 2,6-dimethylbenzoquinone (DMBQ), 2,6-dimethoxybenzoquinone (DMOBQ), and 9,10-phenanthraquinone (PHQ) enhance the rate of H2O2 reduction by ascorbate, under anaerobic conditions, as detected from the amount of methane produced after hydroxyl radical reaction with dimethyl sulfoxide. The amount of methane produced increases with an increase in the quinone one-electron reduction potential. The most active quinone in this series, PHQ, is only 14% less active than the classic Fenton reagent cation, Fe(2+), at the same concentration. Since PHQ is a common toxin present in diesel combustion smoke, the possibility that PHQ-mediated catalysis of hydroxyl radical formation is similar to that of Fe(2+) adds another important pathway to the modes in which PHQ can execute its toxicity. Because quinones are known to enhance the antitumor activity of ascorbate and because ascorbate enhances the formation of H2O2 in tissues, the quinone-mediated reduction of H2O2 should be relevant to this type of antitumor activity, especially under hypoxic conditions.

Roles of hydrophilicities and hydrophobicities of dye and sacrificial electron donor on the photochemical pathway.

Relative rates of the photosensitized production of singlet oxygen ((1)O(2)) and of superoxide (O(2) (•-)) were determined using different couples of dyes and sacrificial electron donors (SEDs) of either high or low hydrophobicities. Such rates were also measured in the absence and presence of single unilamellar vesicles (SUVs) with 9DMPC:1DMPA mol ratio composition. The dyes aluminum phthalocyanine tetrasulfonate (AlPcS(4)) and pheophorbide-a (PHEO) were used as hydrophilic and hydrophobic photosensitizers, respectively. Xanthine (X) and glutathione (GSH) were used as hydrophobic and hydrophilic SEDs, respectively. The presence of SUVs in the aqueous sample produces the physical separation or encounter of SEDs and photosensitizers according to their membrane binding constants. When both the SED and the photosensitizer are localized within the same phase, a strong decrease in the rate of (1)O(2) formation, united to a strong increase in the rate of O(2) (•-) formation, is observed, relative to when both of these species are localized in different phases. The lipid phase is always present in the biological milieu. Thus, the use of a hydrophobic couple of both dye and SED (as in the case of X and PHEO), as well as a hydrophilic couple of both dye and SED (as in the case of GSH and AlPcS4), should strongly favor the Type I mechanism over the Type II. Since only a small number of hydroxyl radicals are needed to initiate a chain reaction of phospholipid peroxidation, the latter could be more toxic to the tumor tissue than peroxidation by a much higher concentration of singlet oxygen molecules.

Role of quinones in the ascorbate reduction rates of S-nitrosoglutathione.

Quinones are one of the largest classes of antitumor agents approved for clinical use, and several antitumor quinones are in various stages of clinical and preclinical development. Many of these are metabolites of, or are, environmental toxins. Because of their chemical structure they are known to enhance electron transfer processes such as ascorbate oxidation and NO reduction. The paraquinones 2,6-dimethyl-1,4-benzoquinone (DMBQ), 1,4-benzoquinone, methyl-1,4-benzoquinone, 2,6-dimethoxy-1,4-benzoquinone, 2-hydroxymethyl-6-methoxy-1,4-benzoquinone, trimethyl-1,4-benzoquinone, tetramethyl-1,4-benzoquinone, and 2,3-dimethoxy-5-methyl-1,4-benzoquinone; the paranaphthoquinones 1,4-naphthoquinone, menadione, 1,4-naphthoquinone-2-sulfonate, 2-ethylsulfanyl-3-methyl-1,4-naphthoquinone and juglone; and phenanthraquinone (PHQ) all enhance the anaerobic rate of ascorbate reduction of GSNO to produce NO and GSH. Rates of this reaction were much larger for p-benzoquinones and PHQ than for p-naphthoquinone derivatives with similar one-electron redox potentials. The quinone DMBQ also enhances the rate of NO production from S-nitrosylated bovine serum albumin upon ascorbate reduction. Density functional theory calculations suggest that stronger interactions between p-benzo- or phenanthrasemiquinones and GSNO than between p-naphthosemiquinones and GSNO are the major causes of these differences. Thus, quinones, and especially p-quinones and PHQ, could act as enhancers of NO release from GSNO in biomedical systems in the presence of ascorbate. Because quinones are exogenous toxins that could enter the human body via a chemotherapeutic application or as an environmental contaminant, they could boost the release of NO from S-nitrosothiol storages in the body in the presence of ascorbate and thus enhance the responses elicited by a sudden increase in NO levels.

Quinone-enhanced reduction of nitric oxide by xanthine/xanthine oxidase.

The quinones 1,4-naphthoquinone, methyl-1,4-naphthoquinone, tetramethyl-1,4-benzoquinone, 2,3-dimethoxy-5-methyl-1,4-benzoquinone, 2,6-dimethylbenzoquinone, 2,6-dimethoxybenzoquinone, and 9,10-phenanthraquinone enhance the rate of nitric oxide reduction by xanthine/xanthine oxidase in nitrogen-saturated phosphate buffer (pH 7.4). Maximum initial rates of NO reduction (V(max)) and the amount of nitrous oxide produced after 5 min of reaction increase with quinone one- and two-electron redox potentials measured in acetonitrile. One of the most active quinones of those studied is 9,10-phenanthraquinone with a V(max) value 10 times larger than that corresponding to the absence of quinone, under the conditions of this work. Because NO production is enhanced under hypoxia and under certain pathological conditions, the observations obtained in this work are very relevant to such conditions.

Quinone-enhanced sonochemical production of nitric oxide from s-nitrosoglutathione.

Sonolysis at 75 kHz of argon- and air-saturated aqueous solutions at pH 7.4 containing s-nitrosogluthathione (GSNO) enhances the production rate of nitric oxide (NO). The quinones, anthraquinone-2-sulfonate (AQ2S) and anthraquinone-2,7-disulfonate (AQ27S) further enhance the NO production over that produced in quinone-depleted sonicated solutions. In contrast, the hydrophobic quinones juglone (JQ) and 1,4-naphthoquinone (NQ) inhibit ultrasound-induced NO detection as compared to quinone-depleted solutions. Larger sonolytical decomposition of the hydrophobic quinones NQ and JQ, as compared to AQ2S and AQ27S, is detected which correlates with a larger production of pyrolysis-derived carbon-centered radicals. Reaction of those radicals with NO could explain NQ and JQ inhibition. This work suggests that sulfonated quinones could be used to enhance NO release from GSNO in tissues undergoing ultrasound irradiation.

Thiols oxidation and covalent binding of BSA by cyclolignanic quinones are enhanced by the magnesium cation.

A novel cyclolignanic quinone, 7-acetyl-3',4'-didemethoxy-3',4'-dioxopodophyllotoxin (CLQ), inhibits topoisomerase II (TOPO II) activity. The extent of this inhibition was greater than that produced by the etoposide quinone (EQ) or etoposide. Glutathione (GSH) reduces EQ and CLQ to their corresponding semiquinones under anaerobic conditions. The latter were detected by EPR spectroscopy in the presence of MgCl(2) but not in its absence. Semiquinone EPR spectra change with quinone/GSH mol ratio, suggesting covalent binding of GSH to the quinones. Quinone-GSH covalent adducts were isolated and identified by ESI-MS. These orthoquinones also react with nucleophilic groups from BSA to bind covalently under anaerobic conditions. BSA thiol consumption and covalent binding by these quinones are enhanced by MgCl(2). Complex formation between the parent quinones and Mg(+2) was also observed. Density functional calculations predict the observed blue-shifts in the absorption spectra peaks and large decreases in the partial negative charge of electrophilic carbons at the quinone ring when the quinones are complexed to Mg(+2). These observations suggest a possible role of Mg(+2) chelation by these quinones in increasing TOPO II thiol and/or amino/imino reactivity with these orthoquinones.

Ortho-quinone-enhanced ascorbate oxidation. Combined roles of lipid charge and the magnesium cation.

Quinones are widely distributed compounds in nature. Of these, ortho-quinones are found to be involved in the pathogenic mechanism of Parkinson's disease, in oxidative deaminations to free-radical redox reactions, and as intermediates in the pathways implicated in the carcinogenicity of 2,3- and 3,4-catechol estrogens. Addition of MgCl(2) to solutions of the hydrophobic ortho-quinones, 1,10-phenanthroquinone (PHQ) and beta-lapachone (LQ) enhances ascorbate oxidation in the absence or presence of large unilamellar vesicles (LUVs) of the neutral lipid dimyristoylphos-phatidylcholine (DMPC), although initial rates of ascorbate oxidation are smaller in the presence of lipid as compared to its absence. Addition of this salt to solutions of the para-quinone 1,4-naphthoquinone (NQ) did not affect the ascorbate rate of oxidation in the absence or presence of DMPC. Addition of MgCl(2) to semiquinone solutions of PHQ or LQ in the presence or absence of DMPC increases semiquinone stability, as detected from the semiquinone disproportionation equilibrium displacement to semiquinone formation. Furthermore, MgCl(2) increases the partition of the ortho-semiquinones into the aqueous phase, although no such effect is observed for the semiquinone of NQ. For all the quinones under study, smaller rates of ascorbate oxidation and of semiquinone equilibrium concentration occur in the presence of negatively charged LUVs composed of an equimolar mixture of DMPC and dimyristoylphosphatidic acid DMPA. Ascorbate oxidation rate enhancements correlate with an increase in semiquinone concentration with addition of MgCl(2), in the absence or presence of neutral lipid. This observation favors the proposition that ascorbate oxidation rate increases are caused by semiquinone thermodynamic stabilization. Thus, the ascorbate oxidation rate enhancement by MgCl(2) in solutions containing hydrophobic ortho-quinones is still possible in systems with hydrophobic environments analogous to that of DMPC.

Sonochemically induced covalent binding of calf thymus DNA by aziridinylquinones.

Sonolysis of argon- or oxygen-containing samples in the presence of calf thymus DNA and the diaziridinylquinones 2,5-bis-aziridin-1-yl-3,6-dichloro-1,4-benzoquinone (AZClQ) and 2,5-bis(carboethoxyamino)-3,6-diaziridinyl-1,4-benzoquinone (AZQ) produced quinone-DNA covalent adducts at pH 5.5 and to a much lesser extent at pH 7.4. The corresponding semiquinone derivatives are detected using EPR spectroscopy after sonolysis of argon-saturated solutions at pH 7.4. The amount of covalent adducts decreases with addition of SOD, indicating a role of superoxide in this process. Addition of oxygen to the purging gas decreased but did not eliminate this covalent adduct. Thus this work suggests a possible synergism between bioreductive quinones and ultrasound in antitumor therapies based on alkylating quinone-DNA adduct formation with potential applications to both hypoxic and normally oxygenated conditions.

Alkaline-earth cations enhance ortho-quinone-catalyzed ascorbate oxidation.

Ortho-quinones 1,10-phenanthroquinone and beta-lapachone but not para-quinones naphthazarin (NZQ) and 1,4-naphthoquinone enhance ascorbate oxidation in the presence of MgCl(2) and CaCl(2) at constant ionic strength. Alkaline-earth cation chelation is observed for the ortho-semiquinones but not for the para-semiquinones, while no interaction between these quinones (with the exception of NZQ) or ascorbate and these salts was detected, suggesting that semiquinone-metal complexes are responsible for the catalytic action on ascorbate oxidation of these metal salts in the presence of these ortho-quinones. Thus, redox cycling efficiency of the quinones under study here, in the presence of ascorbate, depends not only on the quinone redox potential but also on the semiquinone ability to chelate alkaline-earth cations.

Reductive activation and thiol reactivity of benzazolo[3,2-a]quinolinium salts.

Derivatives of benzazolo[3,2-a]quinolium salts (QSDs) are reductively activated by the enzymatic reducing agents hypoxanthine (or xanthine)/xanthine oxidase and NADH dehydrogenase as evidenced by the increase in rates of ferricytochrome c (Cyt(III)c) reduction and oxygen consumption, respectively. No correlation between Michaelis-Menten parameters and QSDs redox potentials was found regarding anaerobic or aerobic Cyt(III)c reduction, although maximum rates were observed for nitro-containing QSDs. However, oxygen consumption rates correlate with QSDs redox potentials when NADH dehydrogenase is used as reducing agent. QSDs bind covalently to bovine serum albumin (BSA) under anaerobic conditions, in the presence, and less in the absence, of HX/XO and only if the nitro group is present at the QSD. QSDs react with glutathione (GSH) in the presence of HX/XO but not in its absence, under anaerobic conditions. The amount of reacted GSH increases, and the relative amount of GSSG formed decreases, with an increase in the QSD reduction potential, thus indicating that GSH reacts with reduced nitro-containing QSDs mainly in a manner which does not involve the production of GSSG, presumably, through the formation of the nitroso-QSD-GSH conjugate. QSDs are, thus, novel nitro-containing heterocyclic compounds which could be bioreductively activated to react with oxygen and thiols.

Múltiples estrategias de evasión de la respuesta inmune por citomegalovirus humano: Mecanismos moleculares / Multiple estrategies of the immune response by human cytomegalovirus. MHC down-regulation

El citomegalovirus humano (HCMV), miembro de la familia de los herpevirus, es un patógeno ubicuo importante en individuos inmunosuprimidos y/o inmunológicamente inmaduros. El balance global en la relación huéspedd-virus depende del estado del sistema inmune, el cual contribuye a través de varios mecanismos efectores al control viral. Después de la primoinfección, aún los individuos inmunocompetentes son incapaces de erradicar al virus debido al establecimiento de un estado de persistencia viral que es resultado del desarrollo de múltiples estrategias de evación al sistema inmune, entre las que se incluyen el ocultamiento viral, el efecto de suspresión inmune y la interferencia a los proceso naturales de presentación de antígeno. Probablemente el mecanismo más relevante para el éxito de la infección viral sea la evasión de la respuesta inmune mediada por células, a través de la disminución de la expresión de moléculas de histocompatibilidad en las células infectadas

Prevalencia de seropositividad a citomegalovirus en pacientes con neoplasias hematológicas / Seroprevalence infection with sytomegalovirus in hematological malignancies patients

La infección por citomegalovirus es ubicua y generalmente asintomática. La reactivación o infección primaria en pacientes con cáncer, particularmente leucemia y linfoma, en individuos con inmunosupresión celular y receptores de transplante, establecen a este virus como importante patógeno en humanos. Con el propósito de determinar la prevalencia de seropositividad a cintomegalovirus en pacientes con neoplasias hematológicas en el Instituto Nacional de Cancerología, se realizó la detección de anticuerpos séricos totales contra citomegalovirus en un grupo de pacientes con este padecimiento. La prevalencia de seropositividad a citomegalovirus fue eleva uniformemente mayor de 85 por ciento en los cuatro grupos de estudio, independiente de sexo y diagnóstico. De los factores estudiados, la edad fue el único que se asoció a la presencia de anticuerpos contra citomegalovirus. Es necesario extender estos resultados, a través de un análisis comparativo en muestras de individuos de otras instituciones con diferentes niveles de ingreso. De esta manera, se podrá obtener mejor conocimiento de la distribución de infección de este virus en la población mexicana.