ABSTRACT
Malignant gliomas are resistant to many kinds of treatments including chemotherapy, radiotherapy and other adjuvant therapies. Autophagy is a novel response of cancer cells to ionizing radiation (IR) or chemotherapy, but its significance and underlying mechanism remains largely elusive. Induction of autophagy in glioma cells using irradiation and arsenic trioxide (ATO) has been reported separately. However, the combined effects of ATO and IR on the cell death processes of malignant glioma cells have not been thoroughly studied, especially in U118-MG cells. In the present study, we investigated the anticancer effect of IR combined with ATO and the underlying mechanisms on U118-MG human malignant glioma cells in vitro. We found that the enhanced cytotoxic effect of IR combined with ATO was through induction of more autophagy in U118-MG cells, which were characterized by the presence of acidic vascular organelle formation, determined by electron microscopic observation and immunoblotting of LC3. Combined treatment could induce more mitotic arrest compared to ATO or IR alone. In addition, we also found that the combined treatment-induced autophagy occurred through inhibition of PI3K/Akt and activation of ERK1/2 signaling pathways. These findings suggest a potential therapeutic strategy for malignant gliomas, which are resistant to various proapoptotic therapies.
Subject(s)
Arsenicals/pharmacology , Autophagy/drug effects , Autophagy/radiation effects , Extracellular Signal-Regulated MAP Kinases/metabolism , MAP Kinase Signaling System/drug effects , Oxides/pharmacology , Phosphatidylinositol 3-Kinases/metabolism , Proto-Oncogene Proteins c-akt/metabolism , Acridine Orange , Apoptosis/drug effects , Apoptosis/radiation effects , Arsenic Trioxide , Cell Line, Tumor , Combined Modality Therapy , Cytoplasmic Vesicles/drug effects , Cytoplasmic Vesicles/radiation effects , Cytoplasmic Vesicles/ultrastructure , Dose-Response Relationship, Radiation , Humans , MAP Kinase Signaling System/radiation effects , Membrane Potential, Mitochondrial/drug effects , Membrane Potential, Mitochondrial/radiation effects , Microscopy, Fluorescence , Microtubule-Associated Proteins/metabolism , Mitosis/drug effects , Mitosis/radiation effects , Mitotic Index , Radiation, Ionizing , Time FactorsABSTRACT
Acridine orange (AO), a weakly basic fluorescent dye, is permeable to plasma and vesicle membranes and preferentially remains in intracellular acidic regions. Using fluorescence microscopy, we observed dynamic changes in AO-loaded cultured malignant melanoma cells during illumination with blue light. Immediately after the start of the illumination, the successive disruption of vesicles was observed as a flash of fluorescence, and shortly after that, blebs were formed on the plasma membrane. These cells died within 5 min. Vesicle disruption was completely inhibited when cells were treated with the vacuolar H(+)-ATPase inhibitor bafilomycin A1 followed by loading with AO, but not when bafilomycin A1 was treated after AO loading. Thus, the filling of AO in the vesicle, which is driven by vacuolar H(+)-ATPase, is initially required for vesicle disruption. In contrast, bafilomycin A1 did not prevent plasma membrane blebbing, indicating that the blebs are formed independently of the vesicle disruption. Acute cell death was inhibited by treatment with bafilomycin A1 before but not after AO loading. Thus, AO- and blue light-induced acute cell death is associated with vesicle disruption rather than bleb formation. Both the vesicle disruption and the formation of plasma membrane blebs were inhibited by removal of oxygen from the cell environment and by singlet oxygen scavengers, sodium azide, ascorbic acid, and L-histidine, but not inhibited by the hydroxyl radical scavenger dimethyl thiourea. Acute cell death was also prevented by singlet oxygen scavengers but not by dimethyl thiourea. Thus, these phenomena are likely caused at least in part by the generation of singlet oxygen. The photosensitive features of plasma and vesicle membranes observed in the present study may be based on the use of the photodynamic effect, such as cancer therapy.
Subject(s)
Cell Membrane/radiation effects , Cytoplasmic Vesicles/radiation effects , Light , Melanoma/radiotherapy , Phototherapy/methods , Acridine Orange , Cell Death/radiation effects , Cell Membrane Permeability/radiation effects , Cells, Cultured , Humans , Melanoma/pathology , Microscopy, Fluorescence , Oxygen/physiology , Vacuolar Proton-Translocating ATPases/physiologyABSTRACT
The p53 protein responds to stress signals by regulating the transcription of a variety of genes. Some of these genes encode secreted proteins that may be involved in the communication between adjacent cells. In this study, a proteomics approach was employed to identify proteins secreted by cells in a p53-dependent manner after DNA damage. In addition to the known transcriptional targets of p53, a set of proteins encoded by genes that are not transcriptional targets of p53 were found to increase in the culture medium after p53 activation. These proteins exit the cell via small, secreted vesicles called exosomes and exosome production by cells was found to be regulated by the p53 response. A p53-regulated gene product, TSAP6, was shown to enhance exosome production in cells undergoing a p53 response to stress. Thus, the p53 pathway regulates the production of exosomes into the medium and these vesicles can communicate with adjacent cells and even cells of the immune system.
Subject(s)
Carcinoma, Non-Small-Cell Lung/metabolism , Cytoplasmic Vesicles/metabolism , Lung Neoplasms/metabolism , Tumor Suppressor Protein p53/physiology , Alleles , Animals , Apoptosis/physiology , Apoptosis/radiation effects , Biomarkers, Tumor/metabolism , Carcinoma, Non-Small-Cell Lung/genetics , Cell Cycle Proteins , Cell Line, Tumor , Cyclophilin A/metabolism , Cytoplasmic Vesicles/radiation effects , DNA Damage/physiology , DNA-Binding Proteins/metabolism , Gene Expression Regulation, Neoplastic , Genes, Tumor Suppressor , HSP90 Heat-Shock Proteins/metabolism , Humans , Lung Neoplasms/genetics , Mice , Mice, Knockout , Oncogene Proteins/biosynthesis , Oncogene Proteins/genetics , Oncogene Proteins/physiology , Oxidoreductases , Peptide Elongation Factor 1/metabolism , Phosphoglycerate Kinase/metabolism , Phosphopyruvate Hydratase/metabolism , RNA, Messenger/genetics , Secretory Rate/radiation effects , Serpins/metabolism , Tumor Suppressor Protein p53/deficiency , Tumor Suppressor Protein p53/genetics , Tumor Suppressor Proteins/metabolismABSTRACT
We previously reported that in preeclampsia Ca-ATPase activity diminishes about 50% in red blood cells, myometrium and syncitiotrophoblast plasma membranes. In this work, we measured the active Ca++ uptake by inside-out vesicles of human red blood cells from preeclamptic and normotensive pregnant women. Active calcium uptake by the vesicles was diminished by 49+/-3% in the preeclamptic women as compared to the gestational controls ( 8.06 +/- 0.11 nmol Ca++/mg protein min, gestational controls; 4.08 +/- 0.1 nmol Ca++/mg protein min, preeclamptics). This lowered calcium uptake correlates well with the lowered Ca-ATPase activity found in the red blood cells ghosts of the preeclamptic women (17.05 +/- 0.96 nmol Pi/mg protein min, gestational controls; 8.85 +/- 0.45 nmol Pi/mg protein min, preeclamptics). The reduced calcium uptake and Ca-ATPase activity of the red cell membranes both appear to be associated with a high level of lipid peroxidation. Thus there is a diminution in the active transport of calcium in the red blood cells of preeclamptic women. If this also occurs in other cell types of the preclamptic women, it could result in an increase in their cytosolic calcium concentration which might be responsible, in part, for some of the symptoms of this disease.
Subject(s)
Calcium/metabolism , Erythrocyte Membrane/metabolism , Lipid Peroxidation/physiology , Pre-Eclampsia/metabolism , Biological Transport, Active , Butylated Hydroxytoluene/pharmacology , Calcium-Transporting ATPases/metabolism , Cytoplasmic Vesicles/drug effects , Cytoplasmic Vesicles/metabolism , Cytoplasmic Vesicles/radiation effects , Erythrocyte Membrane/drug effects , Erythrocyte Membrane/radiation effects , Female , Humans , Pregnancy , Thiobarbituric Acid Reactive Substances/chemistry , Thiobarbituric Acid Reactive Substances/metabolism , Ultraviolet RaysABSTRACT
We approach the problem of an apparently random movement of small cytoplasmic vesicles and its relationship to centrosome functioning. Motion of small vesicles in the cytoplasm of BSC-1 cells was quantified using computer-assisted microscopy. The vesicles move across the cytoplasm frequently changing their directions with negligible net displacement. The autocorrelation function for consecutive velocities of individual vesicles becomes indistinguishable from zero in 10s. Variance in the displacement is proportional to time. The motion of vesicles is anisotropic: It has diffusivity along the radii drawn from the centrosome several times higher than the tangential diffusivity. This anisotropy is abolished by ultraviolet microbeam irradiation of the centrosome when the microtubule array loses radial structure. We conclude that the motion of the vesicles in the cytoplasm can be described as diffusion-like random walk with centrosome-dependent anisotropy. The present analysis quantitatively corroborates the 'trial and error' model of vesicular transport.
Subject(s)
Centrosome/physiology , Cytoplasmic Vesicles/physiology , Epithelial Cells/physiology , Animals , Anisotropy , Cells, Cultured , Centrosome/radiation effects , Chlorocebus aethiops , Cytoplasmic Vesicles/radiation effects , Diffusion , Epithelial Cells/ultrastructure , Kidney/cytology , Ultraviolet RaysABSTRACT
The mechanisms underlying neoplastic epithelial cell killing by ionizing radiation are largely unknown. We discovered a novel response to radiation manifested by autophagy and the development of acidic vesicular organelles (AVO). Acidification of AVO was mediated by the vacuolar H+-ATPase. Staining with the lysosomotropic agent acridine orange enabled us to quantify AVO accumulation and to demonstrate their time- and dose-dependent appearance. The appearance of AVO occurred in the presence of the pan-caspase inhibitor z-Val-Ala-Asp(Ome)-fluoromethyl ketone, but was inhibited by 3-methyladenine, an inhibitor of autophagy. The accretion of AVO in surviving progenies of irradiated cells, and the increased incidence of clonogenic death after inhibition of vacuolar H+-ATPase suggest that formation of acidic organelles represents a novel defense mechanism against radiation damage.