RESUMEN
The adsorption mechanism of linear aliphatic α,ω-dithiols with chain lengths of 6, 8 and 10 carbon atoms on silver and gold nanoparticles has been studied by surface-enhanced Raman scattering (SERS) spectroscopy. SERS spectra provided the structural marker bands of these compounds and they were employed to obtain information about the adsorption and coordination mechanism, the orientation, conformational order, and packing of the aliphatic chains of dithiols on the metal nanoparticle surface. The effect of the type of metal (silver or gold) and the extent of surface coverage on all the above mentioned properties is discussed. It was found that the adsorption of dithiols on Au nanoparticles leads to a more disordered structure of the aliphatic chains of dithiols in comparison with the adsorption on Ag nanoparticles. The interaction through both thiol groups makes the adsorption of dithiols on metal surfaces substantially different from that of monothiols; in particular, the orientation of dithiols is perpendicular, while monothiols adopt a tilted orientation. Dithiols may act as linkers between metal nanoparticles and induce the formation of nanogaps with a controllable interparticle distance. The nanogaps thus formed are able to produce hot spots exhibiting a large intensification of electromagnetic field in these points which has been proved by the observation of intense SERS spectra of dithiols until a concentration of 10(-8) M, corresponding to a large Raman enhancement factor of 5 × 10(6).
Asunto(s)
Oro/química , Nanopartículas del Metal/química , Plata/química , Compuestos de Sulfhidrilo/química , Adsorción , Estructura Molecular , Espectrometría Raman , Propiedades de SuperficieRESUMEN
The in vivo antitumour activity of the natural photosensitizer hypericin was evaluated. C3H/DiSn mice were inoculated with fibrosarcoma G5:1:13 cells. When the tumour reached a volume of 40-80mm3 the mice were intraperitoneally injected with hypericin, either in a single dose (5mg/kg; 1 or 6h before laser irradiation) or two fractionated doses (2.5 mg/kg; 6 and 1 h before irradiation with laser light; 532 nm, 70mW/cm2, 168 J/cm2). All tumours in control groups treated with hypericin alone as well as those irradiated with laser light alone had similar growth rates and none of these tumours regressed spontaneously. Complete remission of tumour in photodynamic therapy (PDT)-treated groups was similar (14-17% single dose vs. 33% fractionated dose), but the fractionated schedule of hypericin dosing was found to be more efficient than the single dose, measured by survival assay (p < 0.05). Our experimental model showed that fractionated administration of hypericin can produce a better therapeutic response than single administration.
Asunto(s)
Antineoplásicos Fitogénicos/farmacología , Hypericum , Perileno/análogos & derivados , Fitoterapia , Animales , Antracenos , Antineoplásicos Fitogénicos/administración & dosificación , Antineoplásicos Fitogénicos/uso terapéutico , Línea Celular Tumoral/efectos de los fármacos , Esquema de Medicación , Fibrosarcoma/tratamiento farmacológico , Inyecciones Intraperitoneales , Luz , Masculino , Ratones , Ratones Endogámicos , Perileno/administración & dosificación , Perileno/farmacología , Perileno/uso terapéutico , Fotoquimioterapia , Fármacos Fotosensibilizantes/administración & dosificación , Fármacos Fotosensibilizantes/farmacología , Fármacos Fotosensibilizantes/uso terapéuticoRESUMEN
Glioblastoma multiforme (GBM) is neoplasm which is resistant to all currently used treatment modalities including surgery, radiation therapy and chemotherapy. Photodynamic therapy (PDT) has been suggested as a novel therapeutical approach to the treatment of malignant gliomas. Here, we attempted to enhance hypericin-induced photocytotoxicity and apoptosis by diazepam, a non-selective ligand of peripheral benzodiazepine receptors (PBR) which seem to play an important role in apoptosis regulation. For the study, we used U-87 MG and U373 MG glioma cell lines and primary cultures of GBM cells prepared from peroperatively obtained tumor specimens. The patients included 7 histologically confirmed GBMs. Colorimetric MTT assay was employed to study the photocytotoxic effects of hypericin and diazepam. Flow cytometry was used to detect apoptosis and assess the proapoptotic effects of diazepam. We found that hypericin upon photoactivation exerts strong cytotoxic effects against U-87 MG and U373 MG cells as well as primary GBM cell cultures. No cytotoxic effect of hypericin was observed under dark conditions. Diazepam inhibited cell growth in U-87 MG cells and primary cultures whereas proliferation of U373 MG cells remained unaffected. When hypericin was combined with diazepam, photocytotoxicity was increased in U-87 MG cells and primary cultures unlike U373 MG cells. Flow cytometric analysis revealed photoactivated hypericin-induced apoptosis in both cell lines. Apoptosis was significantly enhanced by diazepam in U-87 MG cells. However, no such effect was observed in U373 MG cells. In the present study, we showed that photocytotoxic effect of hypericin in glioma cells can be potentiated by diazepam. This effect is underlied by the ability of diazepam to facilitate hypericin-induced apoptosis. This work provides support to performe clinical studies involving diazepam in the antiglioma treatment regimens as an apoptosis-modulating agent.
Asunto(s)
Antineoplásicos/farmacología , Apoptosis/efectos de los fármacos , Neoplasias Encefálicas/patología , Diazepam/farmacología , Moduladores del GABA/farmacología , Glioblastoma/patología , Perileno/análogos & derivados , Adulto , Anciano , Antracenos , Interacciones Farmacológicas , Femenino , Citometría de Flujo , Humanos , Masculino , Persona de Mediana Edad , Perileno/farmacología , Fotoquimioterapia , Células Tumorales CultivadasRESUMEN
Surface-enhanced Raman spectroscopy was employed in this work to study the interaction between the antitumoral drug emodin and human serum albumin (HSA), as well as the influence of fatty acids in this interaction. We demonstrated that the drug/protein interaction can take place through two different binding sites which are probably localized in the IIA and IIIA hydrophobic pockets of HSA and which correspond to Sudlow's I and II binding sites, respectively. The primary interaction site of this drug seems to be site II in the defatted albumin. Fatty acids seem to displace the drug from site II to site I in nondefatted HSA, due to the high affinity of fatty acids for site II. The drug interacts with the protein through its dianionic form in defatted HSA (when placed in the site II) and through its neutral form in the site I of nondefatted albumins.
Asunto(s)
Antineoplásicos/farmacología , Emodina/farmacología , Inhibidores Enzimáticos/farmacología , Albúmina Sérica/farmacología , Espectrometría Raman/métodos , Sitios de Unión , Ácidos Grasos/química , Humanos , Enlace de Hidrógeno , Concentración de Iones de Hidrógeno , Ligandos , Modelos Químicos , Unión Proteica , Rayos UltravioletaRESUMEN
The in vivo antitumour activity of the natural photosensitizer hypericin was evaluated. C3H/DiSn mice inoculated with fibrosarcoma G5:1:13 cells were intraperitoneally or intratumourally injected with hypericin (5 mg/kg) and 2 hours later the mice were locally irradiated with laser light (488 nm, 150 mW/cm2, 180 J/cm2) when the tumour reached volume of 40-80 mm3 (approximately 17 days after inoculation). Tumours treated with hypericin alone as well as those irradiated with laser light alone have similar growth rates and none of these tumours regressed spontaneously. The mean tumour volume in hypericin-PDT treated groups was significantly lower in comparison to that found in the control group 3-5 weeks after the therapy. A higher proportion of animals with tumour volume less than 5-fold of the initial volume has been observed in both hypericin-PDT treated groups. Complete response to PDT has been observed for 44.4% of the animals with intraperitoneally administered hypericin and for 33.3% of the animals with intratumourally administered hypericin. Complete remission occurred in treated lesions with 3 mm or less in height. Hypericin-PDT significantly increased survival. However, no statistically significant difference in survival rate of animals has been found between the intratumoural and the intraperitoneal schedule of administration of hypericin.
Asunto(s)
Antineoplásicos Fitogénicos/uso terapéutico , Fibrosarcoma/tratamiento farmacológico , Perileno/análogos & derivados , Perileno/uso terapéutico , Fotoquimioterapia , Animales , Antracenos , Luz , Masculino , Ratones , Ratones Endogámicos C3H , Ratones Endogámicos , Estructura Molecular , Perileno/administración & dosificación , Perileno/química , Análisis de Supervivencia , Células Tumorales CultivadasRESUMEN
Surface-enhanced Raman spectroscopy, resonance Raman spectroscopy and molecular modeling were employed to study the interaction of hypericin (Hyp) with human (HSA), rat (RSA) and bovine (BSA) serum albumins. The identification of the binding site of Hyp in serum albumins as well as the structural model for Hyp/HSA complex are presented. The interactions mainly reflect: (1) a change of the strength of H bonding at the N1-H site of Trp; (2) a change of the Trp side-chain conformation; (3) a change of the hydrophobicity of the Trp environment; and (4) a formation of an H-bond between the carbonyl group of Hyp and a proton donor in HSA and RSA which leads to a protonated-like carbonyl in Hyp. Our results indicate that Hyp is rigidly bound in IIA subdomain of HSA close to Trp214 (distance 5.12 A between the centers of masses). In the model presented the carbonyl group of Hyp is hydrogen bonded to Asn458. Two other candidates for hydrogen bonds have been identified between the bay-region hydroxyl group of Hyp and the carbonyl group of the Trp214 peptidic link and between the peri-region hydroxyl group of Hyp and the Asn458 carbonyl group. It is shown that the structures of the Hyp/HSA and Hyp/RSA complexes are similar to, and in some aspects different from, those found for the Hyp/BSA complex. The role of aminoacid sequence in the IIA subdomains of HSA, RSA and BSA is discussed to explain the observed differences.
Asunto(s)
Perileno/análogos & derivados , Perileno/química , Perileno/farmacocinética , Fármacos Fotosensibilizantes/química , Fármacos Fotosensibilizantes/farmacocinética , Albúmina Sérica/química , Albúmina Sérica/metabolismo , Animales , Antracenos , Bovinos , Humanos , Enlace de Hidrógeno , Técnicas In Vitro , Modelos Moleculares , Conformación Molecular , Fotoquímica , Ratas , Espectrometría RamanRESUMEN
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
Time-resolved fluorescence and absorption measurements are performed on hypericin complexed with human serum albumin, HSA (1:4, 1:1 and approximately 5:1 hypericin: HSA complexes). Detailed comparisons with hypocrellin A/HSA complexes (1:4 and 1:1) are made. Our results are consistent with the conclusions of previous studies indicating that hypericin binds to HSA by means of a specific hydrogen-bonded interaction between its carbonyl oxygen and the N1-H of the tryptophan residue in the IIA subdomain of HSA. (They also indicate that some hypericin binds nonspecifically to the surface of the protein.) A single-exponential rotational diffusion time of 31 ns is measured for hypericin bound to HSA, indicating that it is very rigidly held. Energy transfer from the tryptophan residue of HSA to hypericin is very efficient and is characterized by a critical distance of 94 A, from which we estimate a time constant for energy transfer of approximately 3 x 10(-15) s. Although it is tightly bound to HSA, hypericin is still capable of executing excited-state intramolecular proton (or hydrogen atom) transfer in the approximately 5:1 complex, albeit to a lesser extent than when it is free in solution. It appears that the proton transfer process is completely impeded in the 1:1 complex. The implications of these results for hypericin (and hypocrellin A) are discussed in terms of the mechanism of intramolecular excited-state proton transfer, the mode of binding to HSA and the light-induced antiviral and antitumor activity.
Asunto(s)
Perileno/análogos & derivados , Quinonas/efectos de la radiación , Antracenos , Transferencia de Energía , Polarización de Fluorescencia , Humanos , Técnicas In Vitro , Sustancias Macromoleculares , Perileno/química , Perileno/efectos de la radiación , Fenol , Fotoquímica , Quinonas/química , Albúmina Sérica Bovina/química , Albúmina Sérica Bovina/efectos de la radiaciónRESUMEN
Hypericin and hypocrellin are potential antiviral and antineoplastic agents with multiple modes of light-induced biological activity connected with a production of singlet oxygen and/or excited-state proton transfer and consequent pH drop formation in the drugs environment. In present work light-induced cytotoxicity of hypericin (1 x 10(-5) - 10(-9) mol) and hypocrellin (1 x 10(-5) - 10(-9) mol) and potentiating effect of omeprazole on human leukemic cell line HL-60 was studied. Under dark condition cultivation none cytotoxicity was observed. The only one exception was hypocrellin in concentration 1 x 10(-5) mol which displayed full cytotoxic effect. However, illumination increased cytotoxic effect of hypericin and hypocrellin, both. Omeprazole, an inhibitor of H+K+-ATPase, has been used for testing the hypothetical pH decreasing effect of hypericin and hypocrellin in their cytotoxic mechanism of action. The results of our experiments have shown that in HL-60 cell line the effect of hypericin and hypocrellin at 1 x 10(-6) mol (both) was significantly potentiated by omeprazole in concentrations 1 x 10(-6) - 10(-9) mol. Our results support the hypothesis that the excited-state proton transfer and the consequent acidification of hypericin and hypocrellin environment could play a role in the biological activity of both agents.
Asunto(s)
Omeprazol/farmacología , Perileno/análogos & derivados , Perileno/farmacología , Fármacos Fotosensibilizantes/farmacología , Quinonas/farmacología , Antracenos , Antineoplásicos/agonistas , Antineoplásicos/farmacología , Antivirales/agonistas , Antivirales/farmacología , Supervivencia Celular/efectos de los fármacos , Sinergismo Farmacológico , Células HL-60 , Humanos , Perileno/agonistas , Fenol , Fármacos Fotosensibilizantes/agonistas , Quinonas/agonistasRESUMEN
Poly(dI-dC) in H2O and D2O solution can undergo different equilibrium geometries which strongly depend on the salt nature and concentration. These structures were studied by classical Raman spectroscopy in order to monitor a hydrogen-deuterium exchange kinetics in 8-CH group in inosine. Spectral and isotopic exchange rate changes depending on NaCl concentration were observed and interpreted on the basis of previously obtained results from resonance and classical Raman spectroscopy studies of poly(dI-dC) and hydrogen-deuterium exchange measurements of different conformations of nucleic acids. It is shown that: i) the Raman spectrum of low-salt poly(dI-dC) corresponds to the right-handed polymer with characteristic bands for B conformation, but the value of the retardation factor of isotopic exchange suggests that this form is not a pure canonical B form and that it contains some portion of the A form, ii) the Raman spectrum of the high-salt poly(dI-dC) corresponds to the right-handed polymer with characteristic bands for both the A and B conformations, iii) the retardation factor of hydrogen deuterium exchange for the high-salt form of poly(dI-dC) is essentially higher than in the low-salt form which indicates a dominant presence of the A form in the high-salt conformation of poly(dI-dC). This leads to the conclusion that the high-salt conformation of poly(dI-dC) is a mixture of A and B forms with the predominant A form.
Asunto(s)
Conformación de Ácido Nucleico , Polidesoxirribonucleótidos/química , Espectrometría Raman , Deuterio , Hidrógeno , Cinética , Cloruro de Sodio , Espectrometría Raman/métodosRESUMEN
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
In this paper we examine the interactions of Calf Thymus DNA and the model polynucleotides poly(dA).poly(dT), poly(dAdT)2 and poly(dG.dC)2 with a group of metalloporphyrins derived from the freebase porphyrin tetrakis(4-N-methylpyridyl)porphine, H2(TMpy-P4), by means of ultraviolet absorption spectroscopy, circular dichroism spectroscopy and microcalorimetry. We have studied the interactions of the copper, cobalt, nickel and zinc derivatives of H2(TMpy-P4) in addition to the free base porphyrin itself. We have found strong evidence for an external self-stacking interaction of the Cu(TMpy-P4) and Zn(TMpy-P4) derivatives with poly(dA).poly(dT) and poly(dAdT)2 even at low concentrations of porphyrin, and all of the porphyrin derivatives studied appear to display such a self-stacking in interaction with poly(dA.dT)2 at sufficiently high ratios of porphyrin to polynucleotide.
Asunto(s)
Calorimetría/métodos , Dicroismo Circular , Metaloporfirinas/metabolismo , Polinucleótidos/metabolismo , Animales , Bovinos , Poli dA-dT/metabolismo , Polidesoxirribonucleótidos/metabolismoRESUMEN
The first resonance Raman scattering observation of the low-frequency (LF) region (below 40 up to 12 cm-1) of DNA motions is presented. Since the concentration of the studied DNA solution was very low (1 mg/ml), the spectra features reflect internal vibrations of the macromolecule. The decomposition of the spectra into Lorentzians clearly indicate three intrahelical DNA modes: the corresponding peaks are located at the frequencies 16, 19, and 23 (+/- 1) cm-1. This result is in agreement with our quasi-continuity model of the LF B-form DNA dynamics (V. Lisy, P. Miskovsky and P. Schreiber, J. Biomol. Struct. Dyn. 13, 707 (1996)). The fit of the experimental frequencies to the theory, using the Genetic Algorithms approach, allowed us to make some conclusions about the model force constants which could be found by independent conformational energy calculations. Possible positions of five lowest-frequency DNA peaks, predicted by the model, are discussed.
Asunto(s)
ADN/química , Algoritmos , Animales , Bovinos , Modelos Moleculares , Espectrometría RamanRESUMEN
The four stranded form of polyriboinosinic acid, or poly(rl), formed under conditions of high ionic strength, has been studied principally by resonance Raman spectroscopy excited in the ultraviolet absorbent band of the hypoxanthine residues. UV Absorption and circular dichroism studies were made, principally in order to verify the presence of the quadruplex form at the low concentrations of poly(rl) used, and a trial experiment with the structural probe Tb3+ was also performed. Experimental evidence is found for highly stacked metastable forms present at low concentrations of polynucleotide, which are destroyed by heating in favor of the two well known forms.
Asunto(s)
Poli I/química , Polinucleótidos/química , Dicroismo Circular , Fluorescencia , Modelos Moleculares , Conformación de Ácido Nucleico , Poli I/metabolismo , Polinucleótidos/metabolismo , Espectrometría Raman , Terbio/química , Terbio/metabolismo , Rayos UltravioletaRESUMEN
A simple quasi-continuity model of low-frequency dynamics of DNA macromolecules is presented. The model is based on the phenomenological theory by Volkov and Kosevich (J. Biomol. Struct. Dyn. 8, 1069 (1991)). We propose improvements to their theory by correcting the model energy and corresponding equations of motion, recalculating the model DNA parameters, and estimating the effects of hydration and counterion binding on the masses of DNA subunits. Comparing the calculated low-frequency vibration spectrum and the results of classical Raman scattering experiments from the literature, the values for the model force constants are determined. These values differ significantly from the estimations made by Volkov and Kosevich. A good quantitative agreement with experiment can be obtained both for the case when the "25 cm-1 mode" is an external one, and when it is considered as an intrahelical mode. Problems connected with the qualitative assignments of the calculated and experimentally observed modes are discussed.
Asunto(s)
ADN/química , Cómputos Matemáticos , Modelos Moleculares , VibraciónRESUMEN
Interactions of the antiretroviral hypericin molecule with polynucleotides, i.e. poly(dG-dC), poly(dA-dT), poly(rG) and poly(rC), have been studied in aqueous solutions by resonance Raman spectroscopy, using an UV excitation wavelength which induces a specific resonance enhancement of spectral band intensities corresponding to proper nucleic base modes of vibration. It is shown that: i) hypericin selectively interacts with the N7 sites of purines, ii) the strength of interaction depends on the polymer structure, and: iii) interaction with guanine is stronger than with adenine molecules.
Asunto(s)
Antivirales/química , Perileno/análogos & derivados , Polinucleótidos/metabolismo , Antracenos , Antivirales/metabolismo , Sitios de Unión , Guanina , Estructura Molecular , Perileno/química , Perileno/metabolismo , Poli C/metabolismo , Poli G/metabolismo , Poli dA-dT/metabolismo , Polidesoxirribonucleótidos/metabolismoRESUMEN
Resonance Raman spectra are reported for poly[dA.dT]2, poly[dA].poly[dT], poly[dG.dC]2, poly[dG].poly[dC], poly[dA.dC].poly[dG.dT] and Calf Thymus DNA in interaction with the porphyrin Cu(TMpy-P4). The spectra were obtained under conditions of low salinity and a ratio of porphyrin molecules to nucleotide base pairs of r = 0.05, with an excitation wavelength of 257 nm. The differences between the spectra in interaction with Cu(TMpy-P4) and those of the unmodified nucleic acids are interpreted in terms of the structural changes imposed by the porphyrin. Two different binding modes have been reported in previous studies, a 5'CG-3' specific intercalative mode and an A-T specific outside binding mode. Our results suggest that this latter mode may show a preference for A-A or T-T sites. Furthermore a transition in structure, while remaining within the B-form family of structures, is indicated for both poly[dA].poly[dT] and poly[dG.dC]2 upon addition of Cu(TMpy-P4).
Asunto(s)
Cobre , Sustancias Intercalantes/química , Metaloporfirinas/química , Nucleótidos/química , Polinucleótidos/química , Espectrometría Raman/métodos , Estructura Molecular , Conformación de Ácido Nucleico , Rayos UltravioletaRESUMEN
Confocal laser microspectrofluorometric measurements on human T47D mammary tumor cells have been performed to assess the intracellular distribution of hypericin within the various cell compartments: cytoplasmic membrane, cytoplasm and nucleus. Confocal fluorescence measurements obtained from microvolumes (approximately 1 micron3) located within the three sites of interest show that, while being primarily located in the cell membrane and cytoplasm after a short-term incubation in a 10(-6) M hypericin-containing culture medium, hypericin actually reaches the inside of the cell nucleus after a long-term incubation (210 min). Moreover, owing to the relative fluorescence quantum yields of hypericin determined in vitro when the molecule interacts with DNA, membrane and protein model systems, it is assumed that there is a significant accumulation of the drug into the cell nucleus. Consequently, the nucleus has to be considered as a possible target for the toxic action of hypericin.