RESUMEN
Cationic 5,10,15,20-tetrakis (1-methyl-4-pyridyl) porphyrin was tested as a delivery agent for oligonucleotides. By using fluorescence microimaging, it has been shown that complexation of the porphyrin to the phosphorothioate analog of dT(15) labeled by rhodamine enabled its nonendocytic penetration into the cell and regular distribution in the cytoplasm and preferentially into the nucleus. Time-resolved microfluorescence spectroscopy revealed that the oligonucleotide integrity was kept. A small fraction of the porphyrin molecules seems to undergo change of the binding mode after internalization, probably due to duplex formation between the oligonucleotide and its cellular target sequences, or due to dissociation of the porphyrin from the oligonucleotide and subsequent interactions in the cellular environment.
Asunto(s)
Oligonucleótidos Antisentido/metabolismo , Porfirinas/metabolismo , Espectrometría de Fluorescencia/métodos , Células 3T3 , Animales , Ratones , Oligonucleótidos Antisentido/químicaRESUMEN
Hypericin, an antiretroviral and antineoplastic agent, seems to have multiple modes of light-induced biological activity connected with the production of single oxygen and/or excited-state proton transfer and a consequent pH drop of pH formation in the hypericin environment. In the present study omeprazole, an inhibitor of H+K+-ATPase, and amiloride, an inhibitor of the Na+/H+ exchanger, have been used for testing the hypothetical pH decreasing effect of hypericin in its antineoplastic action. The results of our experiments have shown that in the HL-60 cell line the effect of hypericin (10(-6) mol.l(-1)) was significantly potentiated by omeprazole and 5'-(N,N-dimethyl)-amiloride. The effect of omeprazole seemed to be less specific than that of 5'-(N,N-dimethyl)-amiloride. Our results support the hypothesis that the excited-state proton transfer and the consequent acidification of the hypericin environment could play a role in the biological activity of hypericin. Moreover, both omeprazole and 5'-(N,N-dimethyl)-amiloride are effective potentiating agents of hypericin cytotoxic effect in the HL60 cell line.
Asunto(s)
Amilorida/análogos & derivados , Antineoplásicos/farmacología , Inhibidores Enzimáticos/farmacología , Omeprazol/farmacología , Perileno/análogos & derivados , Fármacos Sensibilizantes a Radiaciones/farmacología , Amilorida/farmacología , Antracenos , Sinergismo Farmacológico , Células HL-60 , Humanos , Leucemia/tratamiento farmacológico , Leucemia/metabolismo , Perileno/farmacología , Intercambiadores de Sodio-Hidrógeno/antagonistas & inhibidores , ATPasa Intercambiadora de Sodio-Potasio/antagonistas & inhibidoresRESUMEN
The resonance Raman spectra of three oligonucleotides with different lengths containing a specific 5'AG3' target doublet for hypericin - a potent antiretroviral and anticancer photoactive agent, and their 1:1 and 1:2 (oligonucleotide: hypericin) complexes are reported. It is shown that the structural arrangement of the oligonucleotides, their structural stability and the local structural arrangement around the 5'AG3' hypericin target, are the factors which determine the formation of a stable, specifically bounded DNA-hypericin complexes.
Asunto(s)
ADN/química , ADN/metabolismo , Perileno/análogos & derivados , Fármacos Sensibilizantes a Radiaciones/metabolismo , Antracenos , Estructura Molecular , Conformación de Ácido Nucleico , Oligodesoxirribonucleótidos/química , Oligodesoxirribonucleótidos/metabolismo , Perileno/química , Perileno/metabolismo , Fármacos Sensibilizantes a Radiaciones/química , Espectrometría Raman , Relación Estructura-ActividadRESUMEN
Absorption, resonance Raman, surface-enhanced Raman spectroscopy and differential scanning microcalorimetry were employed to study the interaction of hypocrellin A with human serum albumin. The identification of the binding place for hypocrellin A as well as the model for the albumin-hypocrellin A complex are proposed. In this model hypocrellin A interacts with albumin through more than one binding site placed on the protein surface. This model of non-specific interaction could explain why the absorption spectrum of hypocrellin A does not change in the presence of albumin and why the presence of the drug does not change significantly the thermodynamic parameters of the protein, while the Raman spectra show evident changes concerning both the protein and the drug structure. Even if hypocrellin A does not interact with an interior binding site, it can affect deeply the general albumin structure.
Asunto(s)
Antineoplásicos/metabolismo , Antivirales/metabolismo , Perileno/análogos & derivados , Fármacos Fotosensibilizantes/metabolismo , Quinonas/metabolismo , Albúmina Sérica/metabolismo , Antineoplásicos/química , Antivirales/química , Calorimetría , Medicamentos Herbarios Chinos , Humanos , Estructura Molecular , Perileno/química , Perileno/metabolismo , Fenol , Fármacos Fotosensibilizantes/química , Unión Proteica , Quinonas/química , Espectrometría RamanRESUMEN
The fluorescent pH probe carboxy-seminaphtorhodafluor-1 (C-Snarf-1) has been used for laser microspectro-fluorometric assays of intracellular pH in 3T3 mouse fibroblasts treated with hypocrellin A. These results are compared to those previously obtained with the structurally related hydroxylated polycyclic quinone, hypericin (Sureau et al., J. Am. Chem. Soc. 118, 9484-9487, 1996). A mean local intracellular pH drop of 0.6 units has been observed in the presence of 1 microM hypocrellin A after 90 s of exposure to 0.1 microW of laser irradiation at 514.5 nm. The time evolution of the cytoplasm acidification for hypocrellin A-treated cells is faster than that for cells treated by hypericin. Thus, release of protons from an excited state of hypocrellin A appears to be more efficient than that from hypericin. In addition, the pH dependence of the quenching of C-Snarf-1 fluorescence in 3T3 cells under continuous irradiation has been observed. It is shown here that under continuous illumination, a pH decrease is able to induce a modification of the intracellular binding equilibrium of C-Snarf-1 that results in an increase of C-Snarf-1 fluorescence intensity. This latter observation suggests that the protons generated upon the photoexcitation of hypericin or its analogs may be involved in the production of other photoreactive species. Finally, we suggest that, just as for hypericin, this pH drop may be involved in the antiviral and antitumor activity of hypocrellin A.
Asunto(s)
Perileno/análogos & derivados , Fármacos Fotosensibilizantes/farmacología , Quinonas/farmacología , Células 3T3 , Animales , Benzopiranos , Colorantes Fluorescentes , Concentración de Iones de Hidrógeno , Líquido Intracelular/efectos de los fármacos , Líquido Intracelular/metabolismo , Líquido Intracelular/efectos de la radiación , Rayos Láser , Ratones , Naftoles , Perileno/farmacología , Fenol , Fotobiología , RodaminasRESUMEN
The resonance Raman spectra of two oligonucleotides and their complexes with potent antiretrovirally and antineoplastic active photochemical drug hypericin are reported. The Raman spectra of two oligonucleotides containing twelve base pairs on addition of hypericin (one and two molecules per one oligonucleotide) were compared. The first one contains the first nine base pairs of the "rev" gene coming from HIV genome with three base pairs added to stabilize the duplex (5'ATGGCAGGATAT3') and the second one consists of the same content of the nucleotide bases but in changed sequence order which serves as a control sequence (5'ACGTGATGATGA3'). Differences in the spectra of the "rev" gene sequence and control sequence in interaction with the drug indicate that: i) the AG and GA nucleotide doublets are structurally specific targets for hypericin and ii) the hypericin interaction with 5'AG3' target is stronger than with 5'GA3' one.
Asunto(s)
Antineoplásicos/metabolismo , Antivirales/metabolismo , Oligodesoxirribonucleótidos/metabolismo , Perileno/análogos & derivados , Antracenos , Perileno/metabolismo , Espectrometría RamanRESUMEN
Poly(dI-dC) in aqueous solution can undergo different equilibrium geometries which strongly depend on the salt nature and the concentration. These structures were studied by classical Raman spectroscopy (RS). Spectral changes depending on NaCl concentration and on the presence of Ni2+ ions were observed and interpreted on the basis of previously obtained results from resonance RS studies of poly(dI-dC) and classical RS studies for other alternating purine-pyrimidine polydeoxyribonucleotides, i.e. poly(dG-dC), poly(dA-dT) and poly(dA-dC)(dG-dT), which also showed B to Z conformational transitions upon varying the salt concentrations. It is shown that: i) The low-salt structure (0.1 mol/l NaCl) is in the pure canonical B conformation. ii) The high-salt (5 mol/l NaCl) Raman spectrum is similar to that obtained for the low-salt concentration. Thus the high-salt structure corresponds to the right-handed polymer with characteristic bands for both the B (predominant) and A conformations with some weak Z conformation markers which indicate a tendency for B to Z conformational transition of the polymer. iii) The addition of 9.10(-3) mol/l NiCl2 to the high-salt solution induces Z-conformation of the polymer.