RESUMEN
PURPOSE: Oxidative damage and inflammation are expected to be involved in age-related functional decline of lachrymal gland, that induces lachrymal dysfunction; this resulting in dry eye disease. Therefore, we investigated the potential antioxidant effect of 0.2% xanthan gum (XNT) in human corneal epithelial cells (HCE), in comparison with other widely used tear substitute polymers, such as 0.2% hydroxyethylcellulose (HEC), 0.2% hyaluronic acid (HA) and 0.5% carboxymethylcellulose (CMC). METHODS: Subconfluent (80%) HCE (Human Corneal Epithelial) cultures were treated with the different polysaccharides at the above reported concentrations. The effect of every polymer was investigated with and without 0.5 mM H2O2 In detail, hydrogen peroxide was added 1 hour after the addition of polysaccharides. Twelve hours later, reactive oxygen species (ROS) production (dichlorofluorescein diacetate spectrofluorimetric test) was assessed and their values were normalized versus protein content. Morphological analysis was performed by optical microscopy. RESULTS: No morphological differences in HCE compared to control cells (CTRL, cells treated with the buffer used for polymer solubilization) were observed in any of the tested polymers, whereas, in the presence of 0.5 mM H2O2 HCE clearly showed signs of cytotoxicity. Polymers did protect cultures from oxidative stress with XNT>HA = HEC>CMC, as evidenced by microscopic analysis. These results were confirmed from ROS measurements, which showed XNT as the only polysaccharide to restore the levels of ROS comparable to CTRL, in presence of H2O2. CONCLUSIONS: 0.2% xanthan gum was able to protect HCE by oxidative stress, bringing the ROS level down to CTRL values. Considering that in dry eye syndrome oxidative stress sustains inflammation and apoptotic cell death, the use of xanthan gum in ophthalmic preparations could be beneficial.
Asunto(s)
Antioxidantes/farmacología , Epitelio Corneal/efectos de los fármacos , Aditivos Alimentarios/farmacología , Polisacáridos Bacterianos/farmacología , Células Cultivadas , Epitelio Corneal/metabolismo , Humanos , Peróxido de Hidrógeno/toxicidad , Estrés Oxidativo/efectos de los fármacos , Polímeros/farmacología , Especies Reactivas de OxígenoRESUMEN
PURPOSE: The present investigation is aimed to evaluate the effect of a new lipid artificial tear on tear volume and ocular surface signs in a mouse model of dry eye and to test the hypothesis that the combined application with sodium hyaluronate can improve the performance of the treatments. METHODS: A new oil-in-water emulsion, a 0.2% sodium hyaluronate solution, or their combined administration were given to dry eye mice maintained in a controlled environment chamber and treated with scopolamine (0.75-mg transdermal patch). Mice were treated 4 times a day with (a) sodium hyaluronate, (b) emulsion, and (c) sodium hyaluronate followed by emulsion. A control group of mice exposed to controlled environment chamber remained untreated (CTRL+). Tear volume and corneal damage were assessed after 3 and 7 days of treatment by cotton thread test and fluorescein staining. RESULTS: As regards tear volume, sodium hyaluronate did not show a statistically significant effect at either end point; the emulsion was effective after 7 days, whereas their combined administration counteracted the lacrimal decrease induced by the model both at 3 and 7 days. Corneal damage was reduced in all treated groups with respect to CTRL+. This effect was statistically significant after 3 days when the emulsion alone or in combination with sodium hyaluronate was used, while hyaluronate improved this clinical sign after 7 days. CONCLUSIONS: Our data suggest that the new lipid tear substitute can be used to treat clinical signs of dry eye and that the combined administration with hyaluronate can decrease the lag time before the effect, when the evaporative and the aqueous-deficient components are present.
Asunto(s)
Modelos Animales de Enfermedad , Síndromes de Ojo Seco/tratamiento farmacológico , Ácido Hialurónico/administración & dosificación , Lípidos/administración & dosificación , Soluciones Oftálmicas/administración & dosificación , Animales , Córnea/efectos de los fármacos , Lesiones de la Cornea , Combinación de Medicamentos , Síndromes de Ojo Seco/fisiopatología , Emulsiones , Femenino , Ratones , Ratones Endogámicos C57BL , Lágrimas/fisiologíaRESUMEN
Anthocyanins are a class of flavonoids, widely spread throughout the plant kingdom, exhibiting important antioxidant and anti-inflammatory actions as well as chemotherapeutic effects; nonetheless, little is known about the molecular mechanisms by which these activities are exerted. The present study is aimed at investigating molecular mechanisms involved in the chemotherapeutic effects induced by both cyanidin-3-O-beta glucopyranoside (CY3G) and its aglycon form, cyanidin chloride (CY), in human colon cancer cells (CaCo2). The effect on cell growth, reactive oxygen species (ROS) formation and cell cycle/stress proteins modification, including ataxia teleangectasia mutated protein (ATM), p53, p21, 8-oxoguanine DNA glycosylase (OGG1), 70 kDa heat shock protein (HSP70) and topoisomerase IIbeta, as well as on DNA fragmentation, was determined. CY and CY3G treatment affect cell growth and cell proliferation, this latter in a moderately dose-dependent way. Interestingly, ROS level is decreased by any concentration of CY and, only at the lowest concentration, by CY3G. Moreover, the two molecules exert their activities increasing ATM, topoisomerase II, HSP70 and p53 expression. The analysis of DNA fragmentation by Comet assay evidences: (1) a dose-dependent increase in DNA damage only after treatment with CY3G; (2) a more evident trend in the DNA fragmentation when the treatment is performed on agarose embedded cells (cellular atypical Comet); (3) a highly dose-dependent DNA fragmentation induced by CY when the treatment is carried out on agarose embedded naked DNA (acellular atypical Comet). The present findings substantiate a possible chemotherapeutic role of anthocyanins and suggest that CY and CY3G act on CaCo2 by different mechanisms, respectively, ROS-dependent and ROS-independent.
Asunto(s)
Antocianinas/farmacología , Proteínas de Ciclo Celular/metabolismo , Neoplasias del Colon/patología , Daño del ADN , Antineoplásicos , Células CACO-2 , Ciclo Celular/efectos de los fármacos , Proliferación Celular/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Neoplasias del Colon/metabolismo , Ensayo Cometa/métodos , ADN de Neoplasias/genética , Relación Dosis-Respuesta a Droga , Evaluación Preclínica de Medicamentos , Humanos , Proteínas de Neoplasias/metabolismo , Estrés Oxidativo/efectos de los fármacos , Especies Reactivas de Oxígeno/metabolismoRESUMEN
In the Western world cancer is the second leading cause of mortality, and prostate carcinoma represents in men the second most important type of cancer-causing death. We have already shown that resveratrol (200 microM) triggers in DU145, an androgen-resistant prostate cancer cell line, a necrotic-like cell death, while propolis ethanolic extract (100 microg/ml) causes an apoptotic-like cell demise. The present research is aimed to better elucidate the molecular mechanisms activated by the two micronutrients. Vinorelbine bitartrate, a drug widely used in prostate cancer therapy, was utilized as a reference drug, because it is known to induce apoptosis. The combined treatments between the micronutrients and vinorelbine have been studied to test a possible vinorelbine dose reduction, avoiding its side effects without altering its cytotoxic action. In this investigation SEM and TEM analyses were performed to examine the morphological modifications induced; our observations confirmed necrotic cell features after treatment with resveratrol, and apoptotic modifications after propolis. We also measured cell cycle progression to study a correlation with p21 and p53, two well-known cell cycle checkpoints. The levels of HSP27 and HSP70, two chaperones also exerting antioxidant/antiapoptotic functions, were been also analyzed. Our data indicate that the two micronutrients modulate cell cycle distribution, increasing p53 levels, without the induced HSPs being able to rescue DU145 from death. The results presented suggest chemotherapy based on resveratrol and propolis, alone or in combination with vinorelbine, as a potential useful tool for prostate cancer therapy; the increase in cell cycle control and the modulation of HSPs expression reinforce this suggestion.
Asunto(s)
Antineoplásicos Fitogénicos/toxicidad , Própolis/toxicidad , Neoplasias de la Próstata/tratamiento farmacológico , Estilbenos/farmacología , Antineoplásicos Fitogénicos/uso terapéutico , Apoptosis/efectos de los fármacos , Ciclo Celular , Línea Celular Tumoral , Quimioterapia Combinada , Citometría de Flujo , Humanos , Masculino , Microscopía Electrónica , Microscopía Electrónica de Rastreo , Própolis/uso terapéutico , Neoplasias de la Próstata/metabolismo , Neoplasias de la Próstata/patología , Neoplasias de la Próstata/ultraestructura , Resveratrol , Estilbenos/uso terapéuticoRESUMEN
The purpose of this study was to determine in vitro the relationship between ionizing radiation (IR) treatment, reactive oxygen species (ROS) production, lipid peroxidation, glutathione (GSH) levels, and DNA damage of the human benign prostate hyperplasia BPH-1 cell line, and two prostate cancer cell lines, LNCaP, which is androgen-sensitive, and DU-145, which is androgen non-responsive. The cells were analysed after exposure to 1.0 or 2.0 Gy of X-ray radiations. The response to IR treatment was evaluated by examining: ROS production by quantitative analysis with fluorescent probe 5 and 6-carboxy-2'7'-dichlorodihydrofluorescein diacetate bis acetomethyl ester (DCFH-DA), GSH levels by 2,2'-dinitro-5,5'-dithio-benzoic acid (DTNB), and lipoperoxidation by thiobarbituric acid reactive substances (TBARS) analysis. To study IR-induced DNA damage, Single Cell Gel Electrophoresis or comet assay was performed. DU-145 cells were characterized by higher DNA damage, more evident extent of lipid peroxidation, and slighter levels of ROS and GSH compared to BPH-1 or LNCaP. Human benign BPH-1 and cancer LNCaP and DU-145 cell lines are not equal regarding their capability of IR resistance in terms of ROS production, antioxidant potential, IR-induced lipid peroxidation and DNA damage.
Asunto(s)
Neoplasias de la Próstata/radioterapia , Radiación Ionizante , Antioxidantes/farmacología , Línea Celular Tumoral , Ensayo Cometa , ADN/metabolismo , Daño del ADN , Fragmentación del ADN , Ácido Ditionitrobenzoico/farmacología , Relación Dosis-Respuesta en la Radiación , Fluoresceínas/farmacología , Glutatión/metabolismo , Humanos , Peroxidación de Lípido , Masculino , Neoplasias de la Próstata/patología , Especies Reactivas de Oxígeno , Reactivos de Sulfhidrilo/farmacología , Sustancias Reactivas al Ácido Tiobarbitúrico , Rayos XRESUMEN
Rufloxacin belongs to the class of fluoroquinolones that act mainly as specific inhibitors of bacterial Topoisomerase II. These drugs are widely known to be involved in various diseases ranging from cutaneous reactions to aging. The type II photosensitizing activity of Rufloxacin has been already demonstrated on calf thymus DNA and free nucleosides. The aim of this study is to examine in control untreated and UVA irradiated human fibroblasts the modifications on DNA status induced by Rufloxacin added in the culture medium. This allows to investigate the photosensitizing activity of Rufloxacin in a more complex cell model. Fibroblasts, either in the presence or in the absence of Rufloxacin, were exposed to UVA irradiation for different times. An experimental protocol was followed in order to evaluate the amount of single-strand breaks (SSB) and double-strand breaks (DSB) DNA fragmentation by comet assay, and plasmid photocleavage. The presence of oxidized bases was also evaluated using the 8-OH-dGuo test. The comet assay test was also employed to assess cellular repair capacity. The intracellular drug concentration was verified by HPLC-MS. The results confirming the role of Rufloxacin as photosensitizer were: (i) a time-dependent increase in DNA fragmentation when fibroblasts were irradiated in the presence of Rufloxacin; (ii) the efficiency of the cellular repair machinery to be exhaustive after 2 h (whereas no correlation between irradiation time and DNA damage repair was observed with a higher level of DNA fragmentation after shorter irradiation times); (iii) the increased number of cells exhibiting high DNA fragmentation, seen as comets with long tails, was not accompanied by a similar large extent of oxidised DNA base formation, as measured by 8-OH-dGuo analysis; (iv) the double helix SSB, formed in plasmid photosensitization, agreed with the comet assay results, pointing out a good correlation among the cell system and the simpler models used.
Asunto(s)
Desoxiguanosina/análogos & derivados , Fibroblastos/efectos de los fármacos , Fibroblastos/efectos de la radiación , Fluoroquinolonas/farmacología , Fármacos Fotosensibilizantes/farmacología , Quinolonas/farmacología , 8-Hidroxi-2'-Desoxicoguanosina , Biomarcadores/análisis , Células Cultivadas , Ensayo Cometa , Reparación del ADN , Desoxiguanosina/análisis , Rayos UltravioletaRESUMEN
Ochratoxin A (OTA), a mycotoxin produced by Aspergillus ochraceus and other moulds, has recently received growing attention because of its carcinogenic, teratogenic and nephrotoxic properties in both humans and farm animals. Nevertheless, with regard to the mechanism of toxicity, the data in the literature are inconclusive. The aim of our work was to verify in human fibroblasts treated with different OTA dosages the involvement of oxidative pathway in the damage mechanism of this mycotoxin and the possible protective effect exerted by cyanidin 3-O-beta-D-glucoside (C3G), an anthocyanin present in pigmented oranges, red wines, fruits and vegetables. The addition of OTA at 25 and 50 microM concentrations for 48 h determined only a slight but significant (P<.05) increase in radical oxygen species, whereas a substantial increase in their production was observed at longer exposure, in particular, when the fibroblasts were treated with 50 microM OTA for 72 h. Under the same experimental conditions, our data showed a significant (P<.05) increase in the rupture of cellular membrane and high damage to genomic DNA, evaluated by single-cell gel electrophoresis (comet assay), thus confirming the involvement of oxidative stress in the OTA genotoxicity in agreement with other studies. Diversely, mitochondrial functionality does not appear influenced by OTA treatment. C3G (0.125, 0.250 mM) added to the cells treated with 50 microM OTA significantly reduced free radical species production and prevented genomic DNA damage.
Asunto(s)
Antocianinas/farmacología , Daño del ADN , Glucósidos/farmacología , Ocratoxinas/toxicidad , Supervivencia Celular/efectos de los fármacos , Ensayo Cometa , Daño del ADN/efectos de los fármacos , Fibroblastos/efectos de los fármacos , Humanos , Ocratoxinas/antagonistas & inhibidores , Especies Reactivas de Oxígeno/análisis , Factores de TiempoRESUMEN
Vegetables and fruit help the prevention and the therapy of several kinds of cancer because they contain micronutrients, a class of substances that have been shown to exhibit chemopreventive and chemotherapeutic activities. In the present study the effects of resveratrol (100 and 200 microM), a phytoalexin found in grapes, and of the ethanolic extract of propolis (50 and 100 microg/ml), a natural honeybee hive product, were tested in androgen-resistant prostate cancer cells (DU145), a cell line resembling the last stage of prostate carcinoma. A comparison between the activity of these micronutrients and vinorelbine bitartrate (Navelbine), a semi-synthetic drug normally used in the therapy of prostate cancer, was conducted. Several biochemical parameters were tested, such as cell viability (MTT assay), cell membrane integrity (lactate dehydrogenase release), cell redox status (nitric oxide formation, reactive oxygen species production, reduced glutathione levels), genomic DNA fragmentation (COMET assay) with special attention on the presence of apoptotic DNA damage (TUNEL test), and possible mitochondrial transmembrane potential alteration (deltapsi). Our results point out the anticancer activity of resveratrol and propolis extract in human prostate cancer, exerting their cytotoxicity through two different types of cell death: necrosis and apoptosis, respectively. The data obtained suggest the possible use of these micronutrients both in alternative to classic chemotherapy, and in combination with very low dosage of vinorelbine (5 microM).
Asunto(s)
Apoptosis/efectos de los fármacos , Própolis/farmacología , Neoplasias de la Próstata/tratamiento farmacológico , Estilbenos/farmacología , Apoptosis/fisiología , Línea Celular Tumoral , Supervivencia Celular/efectos de los fármacos , Supervivencia Celular/fisiología , Humanos , Masculino , Necrosis , Própolis/uso terapéutico , Neoplasias de la Próstata/metabolismo , Especies Reactivas de Oxígeno/metabolismo , Resveratrol , Estilbenos/uso terapéuticoRESUMEN
The aim of the present research was to determine whether the recently identified and characterized new fibrous amphibole fluoro-edenite may induce a cytopathic response in cultured cells. The final goal was to gain suggestions on the potentiality of fluoro-edenite to be harmful to human beings. Epidemiological studies, in fact, have shown an excess of developing mesothelioma among residents in Biancavilla, a town in eastern Sicily located in the Etna volcanic area. Therefore, we treated human lung fibroblasts, human lung alveolar epithelial cancer cell line A549 and monocyte-macrophage cell line J774 with fluoro-edenite or crocidolite; the latter used as a highly toxic amphibole asbestos reference. Our results show that fluoro-edenite may induce functional modifications and affects some biochemical parameters in tested cell cultures in a concentration and time dependent manner. However, the observed functional modifications induced by fluoro-edenite are generally less dramatic than those induced by crocidolite and more evident on human lung alveolar epithelial cancer cell line A549 with respect to those obtained on human lung fibroblasts or monocyte-macrophage cell line J774. The sequence of the damage is hypothesised to be as follows: at increasing fluoro-edenite concentrations, and/or treatment times, the increase in reactive oxygen species (ROS) production could trigger significant DNA damage in cell cultures, concomitantly with drop in cell metabolism and increase in lactic dehydrogenase release. In conclusion, according to our data, fluoro-edenite appears as a probable carcinogenic agent, responsible for the high incidence of malignant pleural mesothelioma in Biancavilla.
Asunto(s)
Asbestos Anfíboles/toxicidad , Pulmón/efectos de los fármacos , Animales , Asbestos Anfíboles/metabolismo , Asbesto Crocidolita/metabolismo , Asbesto Crocidolita/toxicidad , Línea Celular , Células Cultivadas , Ensayo Cometa , Pruebas Inmunológicas de Citotoxicidad , ADN/efectos de los fármacos , Células Epiteliales/efectos de los fármacos , Células Epiteliales/metabolismo , Fibroblastos/efectos de los fármacos , Fibroblastos/metabolismo , Humanos , Pulmón/citología , Pulmón/metabolismo , Macrófagos/efectos de los fármacos , Macrófagos/metabolismo , Ratones , Óxido Nítrico Sintasa/metabolismo , Óxido Nítrico Sintasa de Tipo II , Especies Reactivas de Oxígeno/metabolismo , Sales de Tetrazolio/metabolismo , Tiazoles/metabolismoRESUMEN
i-NOS and HSP70 antisense oligonucleotides were used to study the role of the two well known stress-regulated molecules on cell survival of both untreated control, and H2O2-stressed human fibroblasts. Cell survival was assessed either by LDH release or by MTT assay. The levels of cytosolic i-NOS and HSP70 were tested by using immunoblotting analysis, and reactive oxygen species (ROS) production was quantified. Compared to the values observed in untreated control cells, anti HSP70-transfected human fibroblasts showed an increase in ROS production, i-NOS level and LDH release. The addition of 0.12 mM H2O2 for 20 min. to the HSP70-deprived fibroblasts did not modify the percentage of LDH release observed in H2O2 stressed cells, but reduced cell viability increasing both ROS production and i-NOS level. Anti i-NOS-transfected fibroblasts, compared to the control untreated cells, showed no modification in ROS production, while cell survival was improved. When treated with H2O2 the i-NOS depleted cells counteracted ROS formation as well as LDH release but negatively affected cell viability and HSP70 levels, compared to the results obtained with H2O2 alone-treated fibroblasts. The data indicates that the induced decrease in HSP70 level in oxidative stress conditions makes fibroblasts more prone to oxidative injury and also increases i-NOS level. Whereas in one way the forced decrease in i-NOS expression seems to counteract ROS production stimulated by the oxidative insult in the cells, in another way, since it causes a decrease in HSP70 expression as well as in cell viability, it seems to activate some unidentified pathways affecting cell demise.
Asunto(s)
Fibroblastos/efectos de los fármacos , Proteínas HSP70 de Choque Térmico/genética , Peróxido de Hidrógeno/farmacología , Óxido Nítrico Sintasa/genética , Oligonucleótidos Antisentido/farmacología , Estrés Oxidativo , Línea Celular Transformada , Supervivencia Celular/efectos de los fármacos , Fibroblastos/metabolismo , Fibroblastos/patología , Formazáns/metabolismo , Proteínas HSP70 de Choque Térmico/metabolismo , Humanos , Peróxido de Hidrógeno/metabolismo , L-Lactato Deshidrogenasa/metabolismo , Óxido Nítrico Sintasa/metabolismo , Óxido Nítrico Sintasa de Tipo II , Especies Reactivas de Oxígeno/metabolismo , Sales de Tetrazolio/metabolismo , TransfecciónRESUMEN
The aim of the present study was to investigate the effects of Levetiracetam, a new antiepileptic drug, on the synthesis of brain-derived neurotrophic factor (BDNF) and inducible nitric oxide synthase (iNOS) in rat cortical astrocyte cultures. The astrocytes were treated for 48 h with different concentrations of Levetiracetam and the expression of BDNF and iNOS was analyzed by immunostaining and immunoblotting analyses. We observed that Levetiracetam is able to stimulate expression of both BDNF and iNOS in a concentration-dependent manner on rat cortical astrocyte cultures. For the BDNF, this effect appears at very low concentrations (1 and 10 microgram/ml), while expression of iNOS appears only at higher dosages (50 microgram/ml). We conclude that Levetiracetam might exert neuroprotective effects, at least in part, via stimulation of neurotrophic factors, thus reducing the extent of inflammation and neuronal death under pathological conditions such as epilepsy.
Asunto(s)
Anticonvulsivantes/farmacología , Astrocitos/enzimología , Factor Neurotrófico Derivado del Encéfalo/metabolismo , Óxido Nítrico Sintasa/metabolismo , Piracetam/análogos & derivados , Piracetam/farmacología , Animales , Astrocitos/citología , Astrocitos/efectos de los fármacos , Células Cultivadas , Inmunohistoquímica , Levetiracetam , Lipopolisacáridos/farmacología , Óxido Nítrico Sintasa de Tipo II , Ratas , Ratas WistarRESUMEN
BACKGROUND: 70 KDa heat shock proteins (HSPs70), either as a constitutive or inducible form, are expressed at very high levels in malignant human tumors of various origin. In different cell types, they are known to play an antiapoptotic role. Resveratrol (3,5,4'-trihydroxy-trans-stilbene), a polyphenol present in red wine, grapes and other dietary and medicinal plants, has been shown to be active in inhibiting multistage carcinogenesis, inducing apoptotic cell death. MATERIALS AND METHODS: With the present study, a possible relationship between HSP70 expression and cell death elicited by resveratrol in DU-145 cells, which mimic the late hormone-refractory stages of prostate carcinoma, was investigated. To this end, we treated DU-145 with different concentrations. (50, 100 and 200 microM) of resveratrol and cell viability, by tetrazolium salts assay (MTT) and membrane breakdown, by lactic dehydrogenase (LDH) release, were measured. The possible induction of oxidative stress was evidenced both by performing a fluorescent analysis of intracellular reactive oxygen species (ROS) production, or evaluating the amount of nitrite/nitrate (NO) in culture medium. In addition, the expression of HSP70 level, evaluated by immunoblotting, was examined and compared with caspase-3 activity (fluorimetrically measured) and DNA damage, determined by Single Cell Gel Electrophoresis or COMET assay. RESULTS: Our data clearly indicate that the addition of resveratrol to DU-145 reduces cell viability and increases membrane breakdown, in a dose-dependent way, without interfering with ROS production or NO synthesis, unless 200 microM resveratrol was added. Furthermore, at low concentration (50-100 microM) resveratrol is able to raise HSP70 levels but, at high concentration (200 microM), the measured levels of protective HSP70 were unmodified with respect to that of the control values. CONCLUSION: Our results confirm the ability of resveratrol to suppress the proliferation of human prostate cancer cells with a typical apoptotic feature, interfering with the expression of HSPs70.