RESUMEN
In this study, cell death regulation and induction in AML cell line from a relapsed MLL-rearranged cell model (MOLM-13) was investigated with doxorubin (Dox) and betulinic acid (BetA), singly and in combination. CyQUANT Direct® and Annexin V/propidium iodide double staining were used to measure the cytotoxic and cell death induction effects of the compounds, respectively. Reactive oxygen species (ROS) generation was measured using 2',7'-dichlorofluorescin diacetate staining. Expressions of proteins and genes were examined by Western blot and reverse transcription polymerase chain reaction analysis, respectively. BetA (20 µM) and Dox (1 µM) indicated a synergistic growth inhibitory effect on MOLM-13 cells. The combined drug caused more cells to reside in irreversible late apoptotic stage compared to the single treatments (p < 0.05). Elevation in ROS may be the synergistic mechanism involved in MOLM-13 cell death since ROS can directly disrupt mitochondrial activity. In contrast, in leukaemic U-937 cells, the combination treatments attenuated Dox-induced cell death. Dox and the drug combination selectively reduced (p < 0.05) a recently reported anti-apoptotic Bcl-2 protein isoform p15-20-Bcl-2 in MOLM-13 by our group, without affecting the usually reported p26-Bcl-2-α. Further studies using known inhibitors of apoptosis are required to confirm the potential of Dox-BetA combination to modulate these pathways.
RESUMEN
The overexpression of antiapoptotic Bcl2 in acute myeloid leukaemia (AML) may contribute to difficulties in eradicating these cells during chemotherapy. In the present study, doxorubicin (Dox) was evaluated for its potential to induce selective apoptotic cell death in AML MOLM13 cells and to modulate autophagy through Bcl2 and Beclin 1 protein expression. Annexin V/propidium iodide and 5(6)carboxyfluorescein diacetate succinimidyl ester (CFSE) flow cytometric analyses were conducted to determine the effects of Dox on cell death and cell proliferation, respectively, following 48 h of coincubation with AML MOLM13 or U937 monocytic cells. The protein expression levels of Bcl2 and Beclin 1 in untreated and treated cells were quantified by western blot analysis. Dox reduced the viability of MOLM13 cells partly by inhibiting cell division and inducing cell apoptosis. Dox demonstrated a level of selectivity in its cytotoxicity against MOLM13 compared to U937 cells (P<0.05). Dox induced a significant decrease in Beclin 1 protein levels in MOLM13 cells without significantly affecting the protein levels in U937 monocytes. A novel Bcl2 1520 kDa (p1520Bcl2) isoform was found to be selectively expressed in AML MOLM13 cells (but absent in the leukaemic cell lines tested, OCIAML2, CML K562 and U937). Dox induced a highly significant inhibition of p1520Bcl2 at concentrations of 0.5, 0.75 and 1 µM (P<0.01). However, the usual 26 kDa Bcl2 (p26Bcl2α) isoform protein expression was not affected by the drug in either the MOLM13 or U937 cells. It was thus postulated that Dox exhibited some selectivity by targeting the p1520Bcl2 isoform in MOLM13 cells and activating Beclin 1 to induce cell death.
Asunto(s)
Beclina-1/metabolismo , Doxorrubicina/farmacología , Leucemia Mieloide Aguda/tratamiento farmacológico , Proteínas Proto-Oncogénicas c-bcl-2/antagonistas & inhibidores , Apoptosis/efectos de los fármacos , Autofagia/efectos de los fármacos , Línea Celular Tumoral , Doxorrubicina/uso terapéutico , Regulación Leucémica de la Expresión Génica , Humanos , Leucemia Mieloide Aguda/patología , Isoformas de Proteínas/antagonistas & inhibidores , Isoformas de Proteínas/metabolismo , Proteínas Proto-Oncogénicas c-bcl-2/metabolismoRESUMEN
Some cancer cells can induce apoptosis in tumour infiltrating cytotoxic T cells as a means of escaping immune destruction. This study examined the expression of the apoptosis-inducing ligands, Fas-L, TRAIL and TNF-alpha, on three representative oral squamous cell carcinoma (OSCC) cell lines, TR146, SCC25 and CAL27 and investigates the contribution of these ligands to tumour cell killing of Jurkat T cells in vitro. All three cell lines were able to induce apoptosis in Jurkat T cells to varying degrees. The TR146 cell line predominantly killed Jurkats via the well known Fas-L/Fas mediated pathway. Although TR146 also expressed low levels of TRAIL and TNF-alpha, these did not contribute significantly to TR146 killing of Jurkats. In contrast, the CAL27 cell line expressed little if any Fas-L but was still able to kill Jurkats effectively via an almost exclusively TRAIL mediated mechanism. The SCC25 cell line expressed significant levels of all three ligands but we were unable to significantly inhibit killing of Jurkats by blocking any one pathway with antibodies. SCC25 may use a combination of mechanisms to kill Jurkats and switch between them to compensate when one mechanism is blocked. We found that stimulation with interferon-gamma (IFN-gamma) induced or increased the expression of apoptosis-inducing ligands on OSCC as well as the killing of Jurkat T cells. Not only did IFN-gamma increase killing of Jurkats, but it changed the contribution of the Fas-L, TRAIL and TNF-alpha mediated mechanisms to the killing of Jurkat T cells by the different cell lines. These mechanisms if reproduced in vivo, could confer survival advantage on OSCC by enabling them to kill tumour invading cytotoxic lymphocytes and evade immune destruction.
Asunto(s)
Carcinoma de Células Escamosas/inmunología , Proteína Ligando Fas/metabolismo , Neoplasias de Cabeza y Cuello/inmunología , Ligando Inductor de Apoptosis Relacionado con TNF/metabolismo , Escape del Tumor/inmunología , Factor de Necrosis Tumoral alfa/metabolismo , Apoptosis/inmunología , Línea Celular Tumoral , Femenino , Citometría de Flujo , Humanos , Células Jurkat , Masculino , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Linfocitos T/inmunologíaRESUMEN
Ultraviolet-A and melanin are implicated in melanoma, but whether melanin in vivo screens or acts as a UVA photosensitiser is debated. Here, we investigate the effect of UVA-irradiation on non-pigmented, lightly and darkly pigmented melanocytes and melanoma cells using electron spin resonance (ESR) spectroscopy. Using the spin trap 5,5 Dimethyl-1-pyrroline N-oxide (DMPO), carbon adducts were detected in all cells. However, higher levels of carbon adducts were detected in lightly pigmented cells than in non-pigmented or darkly pigmented cells. Nevertheless, when melanin levels were artificially increased in lightly pigmented cells by incubation with L-Tyrosine, the levels of carbon adducts decreased significantly. Carbon adducts were also detected in UVA-irradiated melanin-free cell nuclei, DNA-melanin systems, and the nucleoside 2'-deoxyguanosine combined with melanin, whereas they were only weakly detected in irradiated synthetic melanin and not at all in irradiated 2'-deoxyguanosine. The similarity of these carbon adducts suggests they may be derived from nucleic acid- guanine - radicals. These observations suggest that melanin is not consistently a UVA screen against free-radical formation in pigmented cells, but may also act as a photosensitizer for the formation of nucleic acid radicals in addition to superoxide. The findings are important for our understanding of the mechanism of damage caused by the UVA component of sunlight in non-melanoma and melanoma cells, and hence the causes of skin cancer.
Asunto(s)
ADN/química , Radicales Libres/química , Melanocitos/química , Melanoma/química , Carbono/química , Línea Celular Tumoral , Núcleo Celular/efectos de los fármacos , Núcleo Celular/efectos de la radiación , Óxidos N-Cíclicos/farmacología , ADN/efectos de la radiación , Daño del ADN/efectos de la radiación , Nucleótidos de Desoxiguanina/química , Espectroscopía de Resonancia por Spin del Electrón , Humanos , Melanocitos/efectos de la radiación , Melanoma/patología , Melanoma/radioterapia , Rayos Ultravioleta/efectos adversosRESUMEN
The UK's Initial Operational Response (IOR) is a revised process for the medical management of mass casualties potentially contaminated with hazardous materials. A critical element of the IOR is the introduction of immediate, on-scene disrobing and decontamination of casualties to limit the adverse health effects of exposure. Ad hoc cleansing of the skin with dry absorbent materials has previously been identified as a potential means of facilitating emergency decontamination. The purpose of this study was to evaluate the in vitro oil and water absorbency of a range of materials commonly found in the domestic and clinical environments and to determine the effectiveness of a small, but representative selection of such materials in skin decontamination, using an established ex vivo model. Five contaminants were used in the study: methyl salicylate, parathion, diethyl malonate, phorate and potassium cyanide. In vitro measurements of water and oil absorbency did not correlate with ex vivo measurements of skin decontamination. When measured ex vivo, dry decontamination was consistently more effective than a standard wet decontamination method ("rinse-wipe-rinse") for removing liquid contaminants. However, dry decontamination was ineffective against particulate contamination. Collectively, these data confirm that absorbent materials such as wound dressings and tissue paper provide an effective, generic capability for emergency removal of liquid contaminants from the skin surface, but that wet decontamination should be used for non-liquid contaminants.
Asunto(s)
Descontaminación/métodos , Incidentes con Víctimas en Masa , Absorción Cutánea/efectos de los fármacos , Animales , Femenino , Malonatos/toxicidad , Paratión/toxicidad , Forato/toxicidad , Cianuro de Potasio/toxicidad , Salicilatos/toxicidad , Porcinos , Reino UnidoRESUMEN
Breakage of one strand of DNA is the most common form of DNA damage. Most damaged DNA termini require end-processing in preparation for ligation. The importance of this step is highlighted by the association of defects in the 3'-end processing enzyme tyrosyl DNA phosphodiesterase 1 (TDP1) and neurodegeneration and by the cytotoxic induction of protein-linked DNA breaks (PDBs) and oxidized nucleic acid intermediates during chemotherapy and radiotherapy. Although much is known about the repair of PDBs in the nucleus, little is known about this process in the mitochondria. We reveal that TDP1 resolves mitochondrial PDBs (mtPDBs), thereby promoting mitochondrial gene transcription. Overexpression of a toxic form of mitochondrial topoisomerase I (TOP1mt*), which generates excessive mtPDBs, results in a TDP1-dependent compensatory up-regulation of mitochondrial gene transcription. In the absence of TDP1, the imbalance in transcription of mitochondrial- and nuclear-encoded electron transport chain (ETC) subunits results in misassembly of ETC complex III. Bioenergetics profiling further reveals that TDP1 promotes oxidative phosphorylation under both basal and high energy demands. It is known that mitochondrial dysfunction results in free radical leakage and nuclear DNA damage; however, the detection of intermediates of radical damage to DNA is yet to be shown. Consequently, we report an increased accumulation of carbon-centered radicals in cells lacking TDP1, using electron spin resonance spectroscopy. Overexpression of the antioxidant enzyme superoxide dismutase 1 (SOD1) reduces carbon-centered adducts and protects TDP1-deficient cells from oxidative stress. Conversely, overexpression of the amyotrophic lateral sclerosis-associated mutant SOD1G93A leads to marked sensitivity. Whereas Tdp1 knockout mice develop normally, overexpression of SOD1G93A suggests early embryonic lethality. Together, our data show that TDP1 resolves mtPDBs, thereby regulating mitochondrial gene transcription and oxygen consumption by oxidative phosphorylation, thus conferring cellular protection against reactive oxygen species-induced damage.
Asunto(s)
Daño del ADN , ADN Mitocondrial/metabolismo , Proteínas Mitocondriales/metabolismo , Hidrolasas Diéster Fosfóricas/metabolismo , Especies Reactivas de Oxígeno/metabolismo , Transcripción Genética , Animales , ADN Mitocondrial/genética , Ratones , Ratones Noqueados , Proteínas Mitocondriales/genética , Fosforilación Oxidativa , Consumo de Oxígeno , Hidrolasas Diéster Fosfóricas/genéticaRESUMEN
Phosphatidylinositol trisphosphate (PIP3) has been implicated in many platelet functions however many of the mechanisms need clarification. We have used cell permeable analogues of PIP3,1-O-(1,2-di-palmitoyl-sn-glyero-3-O-phosphoryl)-D-myo-inositol-3,4,5-trisphosphate (DiC16-PIP3) or 1-O-(1,2-di-octanoyl-sn-glyero-3-O-phosphoryl)-D-myo-inositol-3,4,5-trisphosphate (DiC8-PIP3) to study their effects on activation on washed human platelets. Addition of either DiC8- or DiC16-PIP3 to human platelets induced aggregation in the presence of extracellular Ca(2+). This was reduced by the presence of indomethacin, the phospholipase C inhibitor U73122 and apyrase. DiC8-PIP3 induced the phosphorylation of Akt-Ser(473) which was reduced by the Akt inhibitor IV, wortmannin and EGTA (suggesting a dependence on Ca(2+) entry). In Fura2 loaded platelets DiC8-PIP3 was effective at increasing intracellular Ca(2+) in a distinct and transient manner that was reduced in the presence of indomethacin, U73122 and 2-aminoethyl diphenylborinate (2APB). Ca(2+) elevation was reduced by the non-SOCE inhibitor LOE908 and also by the SOCE inhibitor BTP2. DiC8-PIP3 induced the release of Ca(2+) from stores which was not affected by the proton dissipating agent bafilomycin A1 and was more potent than the two-pore channel agonist DiC8-PI[3,5]P2 suggesting release from an endoplasmic reticulum type store. DiC8-PIP3 weakly induced the tyrosine phosphorylation of Syk but not of PLCγ2. Finally like thrombin DiC8-PIP3 induced the formation of thromboxane B2 that was inhibited by the Akt inhibitor IV. These studies suggest that PIP3 via Ca(2+) elevation and Akt phosphorylation forms a central role in thromboxane A2 formation and the amplification of platelet activation.
Asunto(s)
Plaquetas/efectos de los fármacos , Calcio/metabolismo , Fosfatos de Fosfatidilinositol/farmacología , Proteínas Proto-Oncogénicas c-akt/metabolismo , Tromboxano A2/metabolismo , Androstadienos/farmacología , Plaquetas/citología , Plaquetas/metabolismo , Ácido Egtácico/farmacología , Ensayo de Inmunoadsorción Enzimática , Fura-2/química , Humanos , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Fosfolipasa C gamma/antagonistas & inhibidores , Fosfolipasa C gamma/metabolismo , Fosforilación/efectos de los fármacos , Agregación Plaquetaria/efectos de los fármacos , Inhibidores de Proteínas Quinasas/farmacología , Proteínas Tirosina Quinasas/metabolismo , Proteínas Proto-Oncogénicas c-akt/antagonistas & inhibidores , Quinasa Syk , Tromboxano A2/análisis , WortmaninaRESUMEN
Skin can be exposed to high-intensity UV-radiation in hot countries and during sunbed use; however, the free-radical damage at these intensities is unknown. We used electron spin resonance spectroscopy to measure free-radical generation in ex vivo human skin/substitutes +/- the spin-trap 5,5 dimethyl-1-pyrroline N-oxide (DMPO) exposed to solar-irradiation equivalent to Mediterranean sunlight. Skin-substitutes, model DNA-photosensitizer systems, lipids and proteins were also irradiated with low-intensity UVA/visible light. Without DMPO a broad singlet was detected (using both irradiations) in skin/substitutes, nail-keratin, tendon-collagen, phospholipid and DNA+melanin or riboflavin. In addition to lipid-derived (tentatively tert-alkoxyl/acyl-) and protein radicals detected with DMPO at lower intensities, isotropic carbon-, additional oxygen- and hydrogen-adducts were detected in solar-irradiated skin/substitutes at higher intensities. Carbon-adducts were detected in UVA-irradiated human skin cells, DNA+melanin or riboflavin and soybean-phospholipid. Anisotropic protein-adducts, comparable to adducts in solar-irradiated tendon-collagen, were absent in UVA-irradiated skin fibroblasts suggesting the trapping of extracellular collagen radicals. Absence of hydrogen-adducts in fibroblasts implies formation in the extracellular compartment. We conclude damage at high intensities is part cellular (carbon- and oxygen-radicals) and part extracellular (protein- and hydrogen/H(+)+e(-) ), and skin substitutes are suitable for sunscreen testing. While UVA absorption and lipid-oxidation is direct, DNA and protein-oxidation require photosensitisation.