RESUMEN
A combination of light, oxygen and a photosensitizer is used to induce death of cancer cells by photodynamic therapy. In this study, we have synthesized several new methyl helianthrone derivatives and compared their phototoxicity with that of hypericin. In contrast to hypericin, methyl helianthrones are soluble in aqueous solutions and have a broad range of light absorbance, which allows the use of polychromatic light. Structural modifications of methyl helianthrone demonstrated that substitution of hydrogen atoms of methyl helianthrone at Positions 2 and 5 with Br atoms or methylation of its phenolic hydroxyls, significantly increases the corresponding singlet oxygen quantum yield and their phototoxicity toward alphaT3-1, M2R and LNCaP cells. The phototoxicity of some of these compounds was similar to that of hypericin. Methyl helianthrones, like hypericin, accumulated mainly in the perinuclear region as evident by confocal microscopy. Irradiation of cells pretreated with methyl helianthrone derivatives generates intracellular reactive oxygen species and lipid free radicals, as shown by a fluorescentic probe and electron paramagnetic resonance methods, respectively. The phototoxicity of these methyl helianthrones as well as their ability to oxidize membrane lipids were significantly decreased on addition of specific Type-II inhibitors, suggesting the involvement of singlet oxygen as the main oxidant.
Asunto(s)
Antracenos/síntesis química , Antracenos/farmacología , Fármacos Fotosensibilizantes/síntesis química , Fármacos Fotosensibilizantes/farmacología , Animales , Antracenos/química , Línea Celular , Línea Celular Tumoral , Supervivencia Celular/efectos de los fármacos , Supervivencia Celular/efectos de la radiación , Ensayos de Selección de Medicamentos Antitumorales , Radicales Libres/metabolismo , Radicales Libres/efectos de la radiación , Histidina/farmacología , Humanos , Luz , Ratones , Estructura Molecular , Perileno/análogos & derivados , Perileno/farmacología , Fotoquimioterapia , Fármacos Fotosensibilizantes/química , Especies Reactivas de Oxígeno/metabolismo , Especies Reactivas de Oxígeno/efectos de la radiación , Oxígeno Singlete/metabolismo , Oxígeno Singlete/efectos de la radiación , Factores de Tiempo , Vitamina E/farmacologíaRESUMEN
Helianthrones 2-4 are a new class of synthetic photosensitizers, which have a molecular skeleton related to that of hypericin. We established that irradiation of heliantrones with visible light leads to the formation of semiquinone radicals and reactive oxygen species. The structures of the paramagnetic anion species produced by electron transfer were calculated on the density functional level and investigated by cyclovoltammetry, UV/vis, and EPR/ENDOR spectroscopy. As with hypericin, the pi system of the helianthrones was found to be considerably deviated from planarity, and, upon electron transfer, deprotonation in the bay region occurs. The structure of the semiquinone radicals was found to be identical in THF, DMF, and aqueous buffered solutions regardless of the means by which reduction was achieved. Semiquinone radicals can be formed via self-electron transfer between the excited state and the ground state or via electron transfer from an electron donor to the excited state of helianthrone. Therefore, the presence of an electron donor significantly enhanced the photogeneration of semiquinone and superoxide radical. The kinetic studies showed that no significant photochemical destruction of helianthrones occurred upon irradiation. Generation of superoxide and singlet oxygen upon irradiation of helianthrones was established by spin trapping techniques. This shows that both type I and type II mechanisms are of importance for the photodynamic action of these compounds.
Asunto(s)
Perileno/análogos & derivados , Fármacos Fotosensibilizantes/química , Electroquímica , Espectroscopía de Resonancia por Spin del Electrón , Cinética , Modelos Moleculares , Conformación Molecular , Oxidación-Reducción , Perileno/química , Fotoquímica , Especies Reactivas de Oxígeno/química , TermodinámicaRESUMEN
Hyperforin is the major active ingredient of Hypericum perforatum (St John's Wort), a traditional antidepressant medication. This study evaluated its inhibitory effects on the synaptic uptake of monoamines in rat forebrain homogenates, comparing the nature of the inhibition at synaptic and vesicular monoamine transporters. A hyperforin-rich extract inhibited with equal potencies the sodium-dependent uptake of the monoamine neurotransmitters serotonin [5-HT], dopamine [DA] and norepinephrine [NE] into rat brain synaptosomes. Hyperforin inhibited the uptake of all three monoamines noncompetitively, in marked contrast with the competitive inhibition exerted by fluoxetine, GBR12909 or desipramine on the uptake of these monoamines. Hyperforin had no inhibitory effect on the binding of [3H]paroxetine, [3H]GBR12935 and [3H]nisoxetine to membrane presynaptic transporters for 5-HT, DA and NE, respectively. The apparent presynaptic inhibition of monoamine uptake could reflect a "reserpine-like mechanism" by which hyperforin induced release of neurotransmitters from synaptic vesicles into the cytoplasm. Thus, we assessed the effects of hyperforin on the vesicular monoamine transporter. Hyperforin inhibited with equal potencies the uptake of the three tritiated monoamines to rat brain synaptic vesicles. Similarly to the synaptosomal uptake, the vesicular uptake was also noncompetitively inhibited by hyperforin. Notably, hyperforin did not affect the direct binding on [3H]dihydrotetrabenazine, a selective vesicular monoamine transporter ligand, to rat forebrain membranes. Our results support the notion that hyperforin interferes with the storage of monoamines in synaptic vesicles, rather than being a selective inhibitor of either synaptic membrane or vesicular monoamine transporters.
Asunto(s)
Antibacterianos/farmacología , Monoaminas Biogénicas/metabolismo , Fluoxetina/análogos & derivados , Inhibidores de la Captación de Neurotransmisores/farmacología , Prosencéfalo/efectos de los fármacos , Sinaptosomas/efectos de los fármacos , Terpenos/farmacología , Animales , Compuestos Bicíclicos con Puentes , Relación Dosis-Respuesta a Droga , Fluoxetina/farmacología , Hypericum/química , Masculino , Floroglucinol/análogos & derivados , Piperazinas/farmacología , Extractos Vegetales/farmacología , Prosencéfalo/metabolismo , Ratas , Sinaptosomas/metabolismoRESUMEN
The internalization mechanism and subcellular distribution of hypericin (Hyp), hypericin tetrasulfonic acid (HypS4) and 1,3,4,6-tetrahydroxyhelianthrone (Hel) were studied in murine colon carcinoma CT26 cells, in protein-free medium or in the presence of serum proteins. The correlation between the extent of uptake of the sensitizers by cells that were incubated in the presence of different serum components, and the internalization mechanisms, was studied. The results indicate that sensitizer internalization may be a result of three mechanisms: partitioning, pinocytosis and endocytosis, and as a direct consequence is targeted to specific subcellular sites. While Hyp and Hel, the two lipophilic sensitizers, were localized in the endoplasmic reticulum after protein-free internalization, the hydrophilic HypS4 was localized in the cytoplasmic membrane and in lysosomes. An endolysosomal internalization route was revealed for Hyp and Hel under serum-enriched conditions showing lysosomal localization, as for HypS4. The lysosomal accumulation of Hyp-serum and specifically Hyp-LDL points to an endocytotic mechanism which is supported by its higher uptake parameter in an LDL-enriched medium, compared to the medium with 10% serum. The different uptake parameters of Hyp to cells, with or without serum, reflect the different mechanisms. Smaller differences in the uptake parameter for HypS4 reflect the distinction between partitioning and endocytosis, which, in this case, are both targeted to the lysosomes. The same uptake parameter of Hel to cells incubated in media with or without serum indicates the absence of the endocytotic mechanism. The interrelationship between subcellular targeting and photodynamic treatment was shown for the three sensitizers Hyp was found to be the most efficient sensitizer for PDT under our illumination protocol and it was dependent on internalization and localization sites.