Asunto(s)
Biopelículas/efectos de los fármacos , Fármacos Fotosensibilizantes/farmacología , Porfirinas/farmacología , Pseudomonas aeruginosa/fisiología , Staphylococcus aureus/fisiología , Biopelículas/efectos de la radiación , Luz , Microscopía Confocal , Microscopía Electrónica de Rastreo , Fármacos Fotosensibilizantes/química , Porfirinas/químicaRESUMEN
The use of endogenous protoporphyrin IX after administration of 5-aminolaevulinic acid (ALA) has led to many applications in photodynamic therapy (PDT). We have previously reported that the conjugation of ALA dendrimers enhances porphyrin synthesis. The first aim of this work was to evaluate the ability of ALA dendrimers carrying 6 and 9 ALA residues (6m-ALA and 9m-ALA) to photosensitise cancer cells. For this aim, we employed LM3 mammary carcinoma cells. In these tumour cells, at low concentrations porphyrin synthesis from dendrimers was higher compared to ALA, whereas at high concentrations, porphyrin synthesis was similar from both compounds. Topical application of ALA dendrimers on the skin overlying a subcutaneous LM3 implanted tumour showed no diffusion of the molecules either to distant skin sites or to the adjacent tumour, suggesting a promising use of the ALA macromolecules in superficial cancer models. As a second objective, we proposed the use of ALA-dendrimers in vascular PDT for the treatment of atherosclerosis. Thus, we focused our studies on ALA-dendrimer's selectivity towards macrophages in comparison with endothelial cells. For this aim we employed Raw 264.7 macrophages and HMEC-1 microvasculature cells. Porphyrin synthesis induced in macrophages by 6m-ALA and 9m-ALA (3 h, 0.025 mM) was 6 and 4.6 times higher respectively compared to the endothelial cell line, demonstrating the high affinity of ALA dendrimers for macrophages. On the other hand, ALA employed at low concentrations was slightly selective (1.7-fold) for macrophages. Inhibition studies suggested that ALA dendrimer uptake in macrophages is mainly mediated by caveloae-mediated endocytosis. Our main conclusion is that in addition to being promising molecules in PDT of superficial cancer, ALA dendrimers may also find applications in vascular PDT, since in vitro they showed selectivity to the macrophage component of the atheromatous plaque, as compared to the vascular endothelium.
Asunto(s)
Adenocarcinoma/tratamiento farmacológico , Ácido Aminolevulínico/farmacología , Dendrímeros/farmacología , Neoplasias Mamarias Animales/tratamiento farmacológico , Fotoquimioterapia/métodos , Fármacos Fotosensibilizantes/farmacología , Ácido Aminolevulínico/química , Animales , Línea Celular Tumoral , Supervivencia Celular/efectos de los fármacos , Supervivencia Celular/fisiología , Dendrímeros/química , Endocitosis/efectos de los fármacos , Endocitosis/fisiología , Células Endoteliales/efectos de los fármacos , Células Endoteliales/metabolismo , Humanos , Macrófagos/efectos de los fármacos , Macrófagos/metabolismo , Masculino , Neoplasias Mamarias Animales/metabolismo , Ratones Endogámicos BALB C , Ratones Desnudos , Microvasos/efectos de los fármacos , Microvasos/metabolismo , Estructura Molecular , Trasplante de Neoplasias , Fármacos Fotosensibilizantes/química , Porfirinas/metabolismo , Piel/efectos de los fármacos , Piel/metabolismoRESUMEN
Photodynamic Therapy (PDT) is an anticancer treatment based on photosensitisation of malignant cells. The precursor of the photosensitiser Protoporphyrin IX, 5-aminolevulinic acid (ALA), has been used for PDT of bladder cancer. Silybin is a flavonoid extracted from Silybum marianum, and it has been reported to increase the efficacy of several anticancer treatments. In the present work, we evaluated the cytotoxicity of the combination of ALA-PDT and silybin in the T24 and MB49 bladder cancer cell lines. MB49 cells were more sensitive to PDT damage, which was correlated with a higher Protoporphyrin IX production from ALA. Employing lethal light doses 50% (LD50) and 75% (LD75) and additional silybin treatment, there was a further increase of toxicity driven by PDT in both cell lines. Using the Chou-Talalay model for drug combination derived from the mass-action law principle, it was possible to identify the effect of the combination as synergic when using LD75, whilst the use of LD50 led to an additive effect on MB49 cells. On the other hand, the drug combination turned out to be nearly additive on T24 cells. Apoptotic cell death is involved both in silybin and PDT cytotoxicity in the MB49 line but there is no apparent correlation with the additive or synergic effect observed on cell viability. On the other hand, we found an enhancement of the PDT-driven impairment of cell migration on both cell lines as a consequence of silybin treatment. Overall, our results suggest that the combination of silybin and ALA-PDT would increase PDT outcome, leading to additive or synergistic effects and possibly impairing the occurrence of metastases.
Asunto(s)
Apoptosis/efectos de los fármacos , Fármacos Fotosensibilizantes/toxicidad , Silimarina/farmacología , Ácido Aminolevulínico/química , Ácido Aminolevulínico/farmacología , Ácido Aminolevulínico/toxicidad , Línea Celular Tumoral , Movimiento Celular/efectos de los fármacos , Sinergismo Farmacológico , Quimioterapia Combinada , Flavonoides/química , Flavonoides/farmacología , Flavonoides/uso terapéutico , Humanos , Luz , Silybum marianum/química , Silybum marianum/metabolismo , Fotoquimioterapia , Fármacos Fotosensibilizantes/química , Fármacos Fotosensibilizantes/uso terapéutico , Silibina , Silimarina/química , Silimarina/uso terapéutico , Neoplasias de la Vejiga Urinaria/tratamiento farmacológico , Neoplasias de la Vejiga Urinaria/metabolismo , Neoplasias de la Vejiga Urinaria/patologíaRESUMEN
The aim of this paper was to investigate a collection of plant extracts from Argentina as a source of new natural photosensitizers (PS) to be used in Photodynamic Inactivation (PDI) of bacteria. A collection of plants were screened for phototoxicity upon the Gram-positive species Staphylococcus epidermidis. Three extracts turned out to be photoactive: Solanum verbascifolium flower, Tecoma stans flower and Cissus verticillata root. Upon exposure to a light dose of 55J/cm(2), they induced 4, 2 and 3logs decrease in bacterial survival, respectively. Photochemical characterisation of S. verbascifolium extract was carried out. PDI reaction was dependent mainly on singlet oxygen and to a lesser extent, on hydroxyl radicals, through type II and I reactions. Photodegradation experiments revealed that the active principle of the extract was not particularly photolabile. It is noticeable that S. verbascifolium -PDI was more efficient under sunlight as compared to artificial light (total eradication vs. 4 logs decrease upon 120min of sunlight). The balance between oxidant and antioxidant compounds is likely to be masking or unmasking potential PS of plant extracts, but employing the crude extract, the level of photoactivity of S. verbascifolium is similar to some artificial PS upon exposure to sunlight, demonstrating that natural resources can be employed in PDI of bacteria.
Asunto(s)
Bacterias Grampositivas/efectos de los fármacos , Fármacos Fotosensibilizantes/farmacología , Extractos Vegetales/farmacología , Bignoniaceae/química , Bignoniaceae/metabolismo , Cissus/química , Cissus/metabolismo , Pruebas Antimicrobianas de Difusión por Disco , Flores/química , Flores/metabolismo , Bacterias Gramnegativas/efectos de los fármacos , Bacterias Gramnegativas/efectos de la radiación , Bacterias Grampositivas/efectos de la radiación , Fotoblanqueo , Fármacos Fotosensibilizantes/química , Extractos Vegetales/química , Raíces de Plantas/química , Raíces de Plantas/metabolismo , Oxígeno Singlete/metabolismo , Solanum/química , Solanum/metabolismoRESUMEN
Photodynamic therapy (PDT) is an anticancer treatment based on light-induced destruction of photosensitised malignant cells. It has been reported that PDT strongly affects cell-cell and cell-substrate adhesion through the reorganization of some cytoskeletal and adhesion proteins. The aim of the present work was to study the changes induced by PDT employing aminolevulinic acid (ALA), on the cytoskeleton actin network and E-cadherin expression. We employed the normal mammary HB4a cell line and its tumor counterpart transfected with the oncogene H-Ras, which has been shown to be resistant to PDT. Ras insertion induces per se disorganization of both F-actin and E-cadherin distribution. ALA-PDT induces on HB4a cells a dramatic disorganization of actin stress fibers, resembling normal Ras-transfected cells. After 48h some features of disorganization remain present. In HB4a-Ras cells, F-actin exhibits signals of photodamage, but distribution is recovered 24h after treatment. On the other hand, PDT did not impact on E-cadherin distribution, other than a transient disorganization, which was recovered at 24h. Moreover, E-cadherin disorganization did not favoured cell-cell detachment after PDT of HB4a-Ras cells. Actin but not E-cadherin constitutes in this model an important target of PDT. The fact that some features of microfilament disorganization remain present in HB4a surviving cells but not in Ras-transfected cells, suggests that cytoskeletal structures such as F-actin may be involved in the mechanisms of resistance to PDT.
Asunto(s)
Actinas/metabolismo , Ácido Aminolevulínico/farmacología , Cadherinas/metabolismo , Fármacos Fotosensibilizantes/farmacología , Proteínas ras/metabolismo , Ácido Aminolevulínico/química , Adhesión Celular/efectos de los fármacos , Adhesión Celular/efectos de la radiación , Línea Celular , Citoesqueleto/efectos de los fármacos , Citoesqueleto/efectos de la radiación , Humanos , Luz , Glándulas Mamarias Humanas/efectos de los fármacos , Glándulas Mamarias Humanas/efectos de la radiación , Fármacos Fotosensibilizantes/química , Transfección , Proteínas ras/genéticaRESUMEN
In the search for possible new anti-cancer agents, we investigated the effects of 75 aqueous and methanol extracts from 41 Argentinean plant species. The effect in cell growth was evaluated in the LM2 mammary adenocarcinoma cells. In a second stage, the highly active selected extracts were assayed in 3 other tumour cell lines: melanoma B16, bladder MB49 and lung A549; and 3 normal cell lines: mammary Hb4a and keratinocytes PAM212 and HaCat. Eight methanol extracts were found to be highly cytotoxic: Collaea argentina leaf, Iochroma australe leaf, Ipomoea bonariensis flower, Jacaranda mimosifolia flower, Solanum amygdalifolium flower, Solanum chacoense leaf, Solanum sisymbriifolium flower and Solanum verbascifolium flower. However, extract inhibition on cell growth was highly dependent on cell type. In general, except for the highly resistant cell lines, the inhibitory concentrations 50% were in the range of 10-150 µg/ml The eight extracts highly inhibited cell growth in a concentration-dependent manner, and in general the methanol extracts were always more active than the aqueous. Murine cells appear to be more sensitive than human cells to the cytotoxic action of the plant extracts. The human melanoma B16 line was the most resistant to four of the extracts. In terms of selectivity, S. verbascifolium was the species which showed most selectivity for tumour cells. Overall, this is one of the first studies focusing on southern South American native plants and their biological effects. Since some species of 5 genera analyzed have been reported to possess different degrees of alkaloid content, we examined microtubule structures after extract treatments. The eight extracts induced destabilization, condensation and aggregation of microtubules in LM2 cells, although no depolarization, typical of Vinca alkaloids damage was observed. In a near future, antitumour activity of purified fractions of the extracts administered at non-toxic doses will be assayed in transplantable murine tumour models.
Asunto(s)
Antineoplásicos/farmacología , Extractos Vegetales/farmacología , Argentina , Línea Celular Tumoral , Supervivencia Celular/efectos de los fármacos , Flores/química , Humanos , Concentración 50 Inhibidora , Ipomoea/química , Lamiaceae/química , Phaseolus/química , Physalis/química , Hojas de la Planta/química , Solanum/química , Tubulina (Proteína)/metabolismoRESUMEN
Photodynamic therapy (PDT) involves the administration of a photosensitizer (PS) followed by illumination with visible light, leading to generation of reactive oxygen species. The mechanisms of resistance to PDT ascribed to the PS may be shared with the general mechanisms of drug resistance, and are related to altered drug uptake and efflux rates or altered intracellular trafficking. As a second step, an increased inactivation of oxygen reactive species is also associated to PDT resistance via antioxidant detoxifying enzymes and activation of heat shock proteins. Induction of stress response genes also occurs after PDT, resulting in modulation of proliferation, cell detachment and inducing survival pathways among other multiple extracellular signalling events. In addition, an increased repair of induced damage to proteins, membranes and occasionally to DNA may happen. PDT-induced tissue hypoxia as a result of vascular damage and photochemical oxygen consumption may also contribute to the appearance of resistant cells. The structure of the PS is believed to be a key point in the development of resistance, being probably related to its particular subcellular localization. Although most of the features have already been described for chemoresistance, in many cases, no cross-resistance between PDT and chemotherapy has been reported. These findings are in line with the enhancement of PDT efficacy by combination with chemotherapy. The study of cross resistance in cells with developed resistance against a particular PS challenged against other PS is also highly complex and comprises different mechanisms. In this review we will classify the different features observed in PDT resistance, leading to a comparison with the mechanisms most commonly found in chemo resistant cells.
Asunto(s)
Resistencia a Medicamentos , Fotoquimioterapia , Fármacos Fotosensibilizantes/farmacología , Animales , Proliferación Celular/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Humanos , Fármacos Fotosensibilizantes/administración & dosificación , Fármacos Fotosensibilizantes/uso terapéutico , Especies Reactivas de Oxígeno/metabolismoRESUMEN
Benzoporphyrin derivative monoacid ring A (BPD-MA) is a second generation hydrophobic photosensitiser for PDT that has been approved for ocular disease treatment. In the present paper we report the results of in vitro studies on the photosensitising activity of Verteporfin (liposomally formulated BPD-MA) using an adenocarcinoma derived cell line. Our findings show a quick and efficient uptake of Verteporfin by LM3 cells, reaching maxima concentrations after 5 hr exposure to 18 microg Verteporfin/ml. Independently on the concentration, plateau levels are attained 5 hr after exposure to Verteporfin. Exposure of the cells to the photosensitiser appears to be safe in the darkness within a broad range of concentrations. The hydroxyl radical scavenger mannitol afforded the highest protection against PDT, while L-tryptophan, a well known and efficient singlet oxygen quencher was not an effective protector at all, showing scavenging activity only when it was supplemented at concentration as high as 10 mM and when 50% of the cells were affected, showing that in addition to singlet oxygen, which is considered the primary cytotoxic agent in PDT, other interconvertible reactive oxygen specie (ROS), in particular HO are also generated. Verteporfin-PDT also induced morphological features typical of apoptotic cells. Results of the present work show that the LM3 adenocarcinoma cell can be effectively sensitised with Verteporfin-PDT.
Asunto(s)
Adenocarcinoma/terapia , Neoplasias Mamarias Animales/terapia , Fármacos Fotosensibilizantes/farmacología , Porfirinas/farmacología , Naranja de Acridina/farmacología , Animales , Apoptosis , Colorantes/farmacología , Relación Dosis-Respuesta a Droga , Relación Dosis-Respuesta en la Radiación , Colorantes Fluorescentes/farmacología , Luz , Ratones , Microscopía Fluorescente , Oxígeno/metabolismo , Especies Reactivas de Oxígeno , Sales de Tetrazolio/farmacología , Tiazoles/farmacología , Factores de Tiempo , Triptófano/farmacología , Células Tumorales Cultivadas , VerteporfinaRESUMEN
The use of more lipophilic derivatives of 5-aminolevulinic acid (ALA) is expected to have better diffusing properties, and after conversion into the parent ALA, to reach a higher protoporphyrin IX (PPIX) formation rate, thus improving the efficacy of topical photodynamic therapy (PDT). Here we have analysed the behaviour of 3 ALA derivatives (ALA methyl-ester, hexyl ester and a 2-sided derivative) regarding PPIX formation, efficiency in photosensitizing cells and mechanism of cellular death. The maximum amount of porphyrins synthesized from 0.6 mM ALA was 47 +/- 8 ng/10(5)cells. The same amount was formed by a concentration 60-fold lower of hexyl-ALA and 2-fold higher of methyl-ALA. The 2-sided derivative failed to produce PPIX accumulation. Applying a 0.6 J cm(-2)light dose, cell viability decreased to 50%. With the 1.5 J cm(-2) light dose, less than 20% of the cells survive, and higher light doses produced nearly total cell killing. Comparing the PPIX production and the induced phototoxicity, the more the amount of porphyrins, the greater the cellular killing, and PPIX formed from either ALA or ALA-esters equally sensitize the cells to photoinactivation. ALA-PDT treated cells exhibited features of apoptosis, independently on the pro-photosensitizer employed. ALA-PDT can be improved with the use of ALA derivatives, reducing the amount of ALA necessary to induce efficient photosensitization.
Asunto(s)
Ácido Aminolevulínico/farmacología , Supervivencia Celular/efectos de los fármacos , Fotoquimioterapia , Fármacos Fotosensibilizantes/farmacología , Animales , Apoptosis/efectos de los fármacos , Ratones , Microscopía Fluorescente , Células Tumorales CultivadasRESUMEN
BACKGROUND: The optimal vehicle to ensure adequate penetration of 5-aminolaevulinic acid (ALA) for its use in photodynamic therapy (PDT) of skin lesions has not been determined. OBJECTIVES: We aimed to study the effects of ALA in various vehicle formulations [saline lotion with and without dimethylsulphoxide (DMSO), cream, liposomes and vaseline] after topical application in a murine subcutaneous adenocarcinoma model. METHODS: The effect of DMSO on porphyrin synthesis and ALA penetration through the skin was studied by measuring the uptake of 14C label from ALA, ALA and porphobilinogen accumulation, and some haem enzyme activities. The tissue distribution and kinetics of porphyrin synthesis after topical application of ALA entrapped in large multilamellar liposomes was also determined. RESULTS: ALA in saline lotion, alone or with 10% DMSO, proved to be the most efficient vehicle for tumour porphyrin accumulation (mean +/- SD 1.75 +/- 0.25 and 2.09 +/- 0.39 microg g-1, respectively), whereas cream and liposomes induced lower levels and identical porphyrin accumulation (0.60 microg g-1). Using ALA + DMSO saline lotion, a higher porphyrin accumulation was found in skin overlying the tumour tissue and in the first 2 mm of tumour, probably due to increased ALA penetration, or greater interconversion to porphyrins, or greater retention of ALA and/or porphyrins. CONCLUSIONS: These findings reinforce the importance of the vehicle in topical ALA-based PDT, and explain the mechanism of action of DMSO in enhancing protoporphyrin IX biosynthesis in superficial lesions.