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1.
Summary The measurement of time correlated intracellular mitochondrial staining with 3,3-dipetyloxacarbocyanine [Di-O-C5(3)] appeared of interest to define the optimal staining conditions. Mitochondrial staining of lymphocytes, monocytes and granulocytes results in different fluorescence signals, related to the numbers of mitochondria, that are present in the cells of these various cell types. Alterations of Di-O-C(5)3 staining in a distinct cell type are due to changes in the physiological or functional state of the mitochondria. It appeared that such alterations occur in cells, which are cultured in the presence of cytosine arabinoside. The effect of cytotoxic drugs upon the mitochondrial membrane potential may be of relevance for the understanding of the mechanism of action, exerted by cytotoxic drugs upon cell biology.In honour of Prof. P. van Duijn 相似文献
2.
Multidrug resistance (MDR) phenotype is characterized by the over-expression of P-glycoprotein (P-gp) on cell plasma membranes that extrudes several drugs out of cells. Cells that express the MDR phenotype are resistant to the mitochondrial related apoptosis and to several anticancer drugs. This study assessed the presence of P-gp in mitochondria and its role in parental drug-sensitive (P5) and in P5-derived MDR1 cells P1(0.5) hepatocellular carcinoma (HCC) cell lines and in drug-sensitive (PSI-2) and mdr1-transfected (PN1A) NIH/3T3 cells. By using Western blot analysis, confocal laser microscopy, measurements of Rhodamine 123 transport across mitochondrial membranes, MDR1 small interfering RNA and flow cytometry analysis, experiments indicate that P-gp is expressed in mitochondria of P1(0.5) and PN1A cells and it is functionally active. Rho 123 accumulation was largely reduced in mitochondria of P1(0.5) cells as compared to those of P5 cells; the reduced uptake of fluorescence in mitochondria of MDR cells was due to P-gp-mediated Rho 123 efflux. In conclusion, these data demonstrate that functionally active P-gp is expressed in the mitochondrial membrane of MDR-positive cells and pumps out anticancer drugs from mitochondria into cytosol. Therefore, P-gp could be involved in the protection of mitochondrial DNA from damage due to antiproliferative drugs. 相似文献
3.
Oxidative stress has been implicated in several pathologies associated with degenerative processes. Mitochondria are involved in cell death by necrosis or apoptosis due to a large load of Ca2+, the formation of reactive oxygen species (ROS), mitochondrial depolarization and the release of cytochrome c that initiates the caspase cascade. Nevertheless, the role of mitochondria in cell death processes induced by hydrogen peroxide (H2O2) has not been fully established. In this study, we analyzed the cytotoxic effect of H2O2 on rho+ human teratocarcinoma (NT2) cells and on mitochondria-DNA depleted rho0 NT2 cells, lacking functional mitochondria. The cells were exposed to H2O2 for 24 h and cell viability was dose-dependently decreased in both cell lines upon H2O2 exposure, although cell susceptibility was higher in rho0 NT2 cells. Moreover a decrease in mitochondrial membrane potential (Deltapsi(m)), mitochondrial cytochrome c release, caspases activation and DNA fragmentation were largely induced by H2O2 and occurred in both cell lines. Nevertheless, increased cell toxicity in rho0 cells upon H2O2 exposure was accompanied by a higher activation of the effector caspases-3 and -6. The data support that, in general, no differences were observed in cells containing functional (rho+) or non-functional (rho0) mitochondria upon H2O2-induced apoptotic cell death. 相似文献
4.
研究丙戊酸钠(sodiumvalproate,VPA)对抗鱼藤酮(Rotenone)诱导的SH-SY5Y细胞损伤的作用及线粒体机制。以l,10μmol/LVPA预处理SH-SY5Y细胞3h,再加入400nmol/LRotenone作用24h。MTT法检测与相差显微镜观察相结合,分析VPA对抗Rotenone损伤的作用;JC-1染色法与Mito-Tracker染色法分析线粒体膜电位及线粒体数量的变化;Clark氧电极法检测细胞呼吸功能;DCFH-DA探针法检测细胞中Ros的含量;并在离体线粒体上观察VPA对Ca^2+诱导的线粒体肿胀的影响。结果发现,1,10p.mol/LVPA预处理SH.SY5Y细胞3h可对抗400nmol/LRotenoneI起的细胞损伤,并且可以提高损伤细胞中线粒体的膜电位,增加线粒体的数量,此外,还可以增强损伤细胞的呼吸功能,降低细胞中ROS的含量,但VPA并不能直接作用于离体的线粒体发挥神经保护作用。由此,VPA具有良好的神经保护作用,其机制与增强线粒体功能和数量、从而改善细胞功能有关,这为其应用于帕金森病的预防与治疗提供了实验依据。 相似文献
5.
We have examined the effect of ethacrynic acid on mitochondrial morphology and distribution as well as on cellular toxicity in cultured human fibroblasts, African Green Monkey B-SC-1 kidney cells, and Chinese hamster ovary cells. Treatment of the above cells with 66 μM ethacrynic acid causes no reduction in cell viability after 2 h but is cytotoxic upon prolonged (6–7 days) exposure. Ethacrynic acid treatment for up to 2 h is found to cause novel shape changes and redistribution of mitochondria, as assessed by immunofluorescence and electron microscopy. Early effects include the transient formation of a mitochondrial reticulum involving the majority of mitochondria, and these reticula are aligned along microtubules. At later times within 2 h, mitochondrial distributions become disoriented (show no association with microtubules), and an aggregation and final positioning of mitochondria around the nucleus is observed. Whole mount electron microscopy shows that mitochondria in treated cells increase in length and form junctions, indicating reticula result from mitochondrial fusion. Electron microscopy of sections through ethacrynic acid induced reticula demonstrates structural continuity in mitochondria at branch points and the presence of regular cristae. Staining of endoplasmic reticulum and mitochondria in intact cells with the cyanine dye 3,3′-dihexyloxacarbocyanine iodide provides evidence of concurrent aggregation of endoplasmic reticulum. Rhodamine 123 staining of living cells followed by immunofluorescent labeling of mitochondria in the same cells indicates that all mitochondria retain a transmembrane potential during the druginduced shape changes and redistributions. The described effects of ethacrynic acid on mitochondrial morphology as well as on cellular toxicity are completely prevented by 0.5 mM dithiothreitol, indicating that ethacrynic acid is acting as a sulfhydryl reagent to produce the observed effects. The above observations also indicate that ethacrynic acid effects on mitochondrial morphology are an early event in the drug-induced cytotoxicity. The generation of varied mitochondrial morphologies by fusion and fission of mitochondria and its modulation by agents such as ethacrynic acid are discussed. © 1994 wiley-Liss, Inc. 相似文献
6.
In order to gain insights into the mechanism of phototoxicity of the neuroleptic drugs fluphenazine, perphenazine and thioridazine in cultured cells, studies were performed with murine 3T3 fibroblasts, aimed at identifying some cellular targets responsible for photoinduced cell death and possible cytotoxic reactive species involved in the photosensitization process. 3T3 fibroblasts incubated with 5 microM drugs and irradiated with UVA light (up to 8 J/cm2) underwent cell death, the extent of which depended on light dose. Of the three drugs, fluphenazine exhibited the highest phototoxicity and 100% cell death was achieved with a light dose of 5 J/cm2. Superoxide dismutase and alpha-tocopherol exerted a dose-dependent protective effect against drug phototoxicity, whereas N-acetylcysteine failed to do so. These findings indicate that superoxide anion and other free radical intermediates, generated in lipophilic cellular environments, play a role in photoinduced toxicity. Phototreatment of drug-loaded cells induces release of the cytosolic enzyme lactate dehydrogenase and causes loss of activity of mitochondrial NADH dehydrogenase, indicating that plasma membrane and mitochondria are among the targets of the phototoxicity of these drugs. 相似文献
7.
Mitochondria are cellular organelles that perform pivotal functions essential for ATP production, homeostasis, and metabolism. Moreover, mitochondria are integral to a variety of cell death and survival pathways. These roles identify mitochondria as a potential target for drugs to treat metabolic and hyperproliferative diseases. Differences in the redox state of pathogenic versus non-pathogenic cells may be exploited to achieve selective anti-proliferative and cytotoxic activity against target cell populations. Pro-oxidant drugs, such as Trisenoxtrade mark and Elesclomoltrade mark, are demonstrating clinical utility in the treatment of cancer. Results obtained with Bz-423 in mice demonstrate the potential for mitochondria-targeted drugs to control disorders of immune function. Research associating an elevated oxidant state with mitochondrial damage, degenerative disease, and aging dictates the need for a better understanding of when and how pharmacological manipulation of mitochondrial function provides most therapeutic benefit. 相似文献
8.
The ovarian cancer cell lines A2780 (wild-type p53) and NIHOVCAR3 (mutated p53) showed, respectively, sensitivity and resistance towards several chemotherapy drugs. We hypothesized that the two cell lines differ in their ability to activate the intrinsic death pathway and have, therefore, dissected the lysosome-mitochondrion signalling pathway by pharmacological inhibition or genetic manipulation of key regulators and executioners. Biochemical and morphological confocal fluorescence studies showed that: (1) In A2780 cells bcl-2 is expressed at an undetectable level, whereas Bax is expressed at a rather high level; by contrast, bcl-2 is highly expressed and Bax is expressed at extremely low levels in NIHOVCAR3 cells; (2) Chemotherapy treatment reduced the expression of bcl-2 in NIHOVCAR3 cells, yet these cells resisted to drug toxicity; (3) Cathepsin D (CD), not cathepsin B or L, mediates the activation of the mitochondrial intrinsic death pathway in A2780 cells; (4) Lysosome leakage and cytosolic relocation of CD occurs in the chemosensitive A2780 cells, not in the chemoresistant NIHOVCAR3 cells; (5) Bax is essential for the permeabilization of both lysosomes and mitochondria in A2780 cells exposed to chemotherapy drugs; (6) CD activity is mandatory for the oligomerization of Bax on both mitochondrial and lysosomal membranes; (7) Bax activation did not occur in the resistant NIHOVCAR3 cells despite their high content in CD. The present data are consistent with a model in which on treatment with a cytotoxic drug the activation of a CD-Bax loop leads to the generalized permeabilization of lysosomes and eventually of mitochondria, thus reaching the point of no return, and culminates with the activation of the caspase cascade. Our data also imply that dysfunctional permeabilization of lysosomes contributes to the development of chemoresistance. 相似文献
9.
Cardiolipin, a polyunsaturated acidic phospholipid, is found exclusively in bacterial and mitochondrial membranes where it is intimately associated with the enzyme complexes of the respiratory chain. Cardiolipin structure and concentration are central to the function of these enzyme complexes and damage to the phospholipid may have consequences for mitochondrial function. The fluorescent dye, 10 nonyl acridine orange (NAO), has been shown to bind cardiolipin in vitro and is frequently used as a stain in living cells to assay cardiolipin content. Additionally, NAO staining has been used to measure the mitochondrial content of cells as dye binding to mitochondria is reportedly independent of the membrane potential. We used confocal microscopy to examine the properties of NAO in cortical astrocytes, neonatal cardiomyocytes and in isolated brain mitochondria. We show that NAO, a lipophilic cation, stained mitochondria selectively. However, the accumulation of the dye was clearly dependent upon the mitochondrial membrane potential and depolarisation of mitochondria induced a redistribution of dye. Moreover, depolarisation of mitochondria prior to NAO staining also resulted in a reduced NAO signal. These observations demonstrate that loading and retention of NAO is dependant upon membrane potential, and that the dye cannot be used as an assay of either cardiolipin or mitochondrial mass in living cells. 相似文献
10.
This research focused on the induction of cytotoxic effects by danthron, a natural anthraquinone derivative on C6 rat glioma cells through exploring the means of cell death and the effects on mitochondrial function. We found that danthron decreased the percentage of viable C6 cells and induced cell morphological changes in a dose-and time-dependent manner. The morphological and nuclei changes (DAPI staining) in C6 cells were observed using a contrast-microscope and fluorescence microscopy, respectively. The results suggest that cell death of C6 cells which are induced by danthron is closely related to apoptotic death. Danthron decreased the level of mitochondrial membrane potential (ΔΨ( m )), stimulated the release of cytochrome c from mitochondria to cytosol and promoted the levels of caspase-9 and caspase-3, or induced the release of AIF and Endo G from mitochondria. Based on both observations, we suggest that the danthron-provoked apoptotic death of C6 cells is mediated through the mitochondria-dependent pathway. Furthermore, our results also indicated that danthron triggered apoptosis through reactive oxygen species (ROS) production which were increased after 1 h exposure of danthron, which was reversed by the ROS scavenger N-acetyl-L: -cysteine (NAC). As a consequence, danthron-mediated cell death of C6 cells via ROS production, mitochondrial transmembrane potential collapse and releases of cytochrome c, AIF and Endo G. Taken together, danthron was demonstrated to be effective in killing C6 rat glioma cells via the ROS-promoted and mitochondria-dependent apoptotic pathways. 相似文献
11.
Changes in the mitochondria of aerobically grown Saccharomyces cerevisiae cells upon deaeration and subsequent aeration of the medium were studied. 1. It is shown that removal of oxygen at the end of the exponential phase of growth (after completion of mitochondria formation) causes a decrease in activity of the respiratory enzymes. The activity of the complete respiratory system decreases much more rapidly than the activities of its fragments (NADH: ferricyanide reductase, succinate:ferricyanide reductase, NADH:cytochrome c reductase, succinate:cytochrome c reductase and cytochrome oxidase). The activities are restored to their initial level upon aeration of the cell suspension. The addition of Tween-80 and ergosterol to the medium prior to deaeration does not prevent inactivation of the respiratory system. All the changes in mitochondria described occurred under conditions where cell division was insignificant. 2. Deaeration of the medium decreases the content of cytochromes b and aa3 in the mitochondrial fraction, cytochrome aa3 “disappearing” more quickly. The concentration of cytochromes in this fraction increases upon subsequent aeration of the cells. The total cytochromal content of the cells remains practically unchanged under the same conditions. 3. According to electron microscopic data, anaerobiosis causes a certain disorganization of mitochondrial cristal membranes. The mitochondrial structures are recovered upon aeration of the yeast cell suspension. It may be reasoned that inactivation and reactivation of the respiratory system are associated with reversible changes in mitochondrial membrane structure. 4. The effect of protein synthesis inhibitors on the restoration of mitochondria was investigated. It is shown that chloramphenicol does not suppress this process. In the presence of cycloheximide, oxygen induces reactivation of the respiratory system and simultaneously the appearance of particles resembling mitochondria. However, these particles gradually undergo morphological changes and the respiratory activity of the mitochondrial fraction decreases. Cycloheximide added to yeast cells that had not been deaerated, did not affect their mitochondria. 5. The results described suggest that the functions of oxygen in the formation of mitochondria are not restricted to the induction of mitochondrial protein synthesis and to the participation in the synthesis of certain non protein membrane components. Evidently, oxygen has a direct effect on the assembly of the respiratory system and mitochondrial membranes as a whole. 相似文献
12.
Glioblastoma is a highly heterogeneous aggressive primary brain tumor, with the glioma stem‐like cells (GSC) being more sensitive to cytotoxic lymphocyte‐mediated killing than glioma differentiated cells (GDC). However, the mechanism behind this higher sensitivity is unclear. Here, we found that the mitochondrial morphology of GSCs modulates the ER–mitochondria contacts that regulate the surface expression of sialylated glycans and their recognition by cytotoxic T lymphocytes and natural killer cells. GSCs displayed diminished ER–mitochondria contacts compared to GDCs. Forced ER–mitochondria contacts in GSCs increased their cell surface expression of sialylated glycans and reduced their susceptibility to cytotoxic lymphocytes. Therefore, mitochondrial morphology and dynamism dictate the ER–mitochondria contacts in order to regulate the surface expression of certain glycans and thus play a role in GSC recognition and elimination by immune effector cells. Targeting the mitochondrial morphology, dynamism, and contacts with the ER could be an innovative strategy to deplete the cancer stem cell compartment to successfully treat glioblastoma. 相似文献
13.
Summary Dimorphic yeast Trigonopsis variabilis is a unique species that can form either an ellipsoidal or a triangular cell depending upon nutritional conditions. This fluorescence microscopic study was intended to correlate morphological changes of mitochondria in the triangular cells with the distribution of the cytoskeleton. In addition, unique features in the behavior of the cytoskeleton were also examined during triangular cell formation. In log-phase cells stained with 4,6-diamidino-2-phenylindole, mitochondrial nucleoids appeared as a string of beads throughout the vegetative growth. The profile of mitochondria stained by 3,3-dihexyloxacarbocyanine iodide showed a network corresponding to the fluorescence images of mitochondrial nucleoids in both mother and daughter cells. Cell-cycle-dependent fragmentation of mitochondria was not discerned. As the culture reached stationary phase, a network of mitochondria gradually changed to form unique rings that were located near the angles of triangular cells. When examined by immunofluorescence microscopy with anti-tubulin antibody, microtubules were found to be well developed along the sides of cells in the cytoplasm of T. variabilis interphase cells. Although distributions of microtubules and mitochondria are different during cell cycle as a whole, cytoplasmic microtubules frequently extended along a part of the mitochondria in budded cells, suggesting correlation of microtubules and mitochondria. Rhodamine-phalloidin staining revealed both actin patches and cables. Actin cables elongated from mother cells into the buds and showed close proximity to mitochondria, although complete overlapping of both structures was rare. Moreover, actin patches localized on the mitochondrial network at a frequency of 65%. These results suggested that actin cables and patches, as well as microtubules, participated in the distribution of mitochondria. The localization of actin patches separated towards opposite ends at a bud tip when the bud grew to medium size. The unique localization of actin patches is responsible for bi-directional growth of the bud, forming triangular cells. 相似文献
14.
Mitochondria in cells of Catharanthus roseus (L.) G. Don in synchronous cell division cultures were observed by double staining using fluorescence microscopy. The cells
were stained with 4′-6-diamidino-2-phenylindole (DAPI) first and subsequently stained with rhodamine 123 (r-123). Immediately
after staining with r-123, yellowishgreen, elongated and moving mitochondria were observed upon excitation at 485 nm. When
the excitation filters were replaced by a UV filter (360 nm), 1 to 7 mitochondrial nucleoids were visible in each mitochondrion
in the same field. Changes in the lengths of mitochondria during the cell cycle obtained from the observations under fluorescence
microscopy by this staining method suggest the occurrence of multiplication of mitochondria concurrent with the cell cycle
of C. roseus. 相似文献
15.
The study has demonstrated that dopamine induces membrane depolarization and a loss of phosphorylation capacity in dose-dependent manner in isolated rat brain mitochondria during extended in vitro incubation and the phenomena are not prevented by oxyradical scavengers or metal chelators. Dopamine effects on brain mitochondria are, however, markedly prevented by reduced glutathione and N-acetyl cysteine and promoted by tyrosinase present in the incubation medium. The results imply that quinone oxidation products of dopamine are involved in mitochondrial damage under this condition. When PC12 cells are exposed to dopamine in varying concentrations (100-400μM) for up to 24h, a pronounced impairment of mitochondrial bio-energetic functions at several levels is observed along with a significant (nearly 40%) loss of cell viability with features of apoptotic nuclear changes and increased activities of caspase 3 and caspase 9 and all these effects of dopamine are remarkably prevented by N-acetyl cysteine. N-acetyl cysteine also blocks nearly completely the dopamine induced increase in reactive oxygen species production and the formation of quinoprotein adducts in mitochondrial fraction within PC12 cells and also the accumulation of quinone products in the culture medium. Clorgyline, an inhibitor of MAO-A, markedly decreases the formation of reactive oxygen species in PC12 cells upon dopamine exposure but has only mild protective actions against quinoprotein adduct formation, mitochondrial dysfunctions, cell death and caspase activation induced by dopamine. The results have indicated that quinone oxidation products and not reactive oxygen species are primarily involved in cytotoxic effects of dopamine and the mitochondrial impairment plays a central role in the latter process. The data have clear implications in the pathogenesis of Parkinson's disease. 相似文献
16.
The diaminobenzidine (DAB) technique for the ultrastructural localization of sites of cytochrome c oxidase activity in animal tissues has been adapted to the visualization of mitochondria in animal cells growing in culture. The modified technique allows the staining of mitochondria in all cells in coverslip preparatins for light microscopy. Electron microscopy of thin sections of material treated by this method has revealed that all mitochondrial profiles within a cell (and only these) are stained and they exhibit a well preserved size and internal structure. Coverslip cultures of synchronized and unsynchronized HeLa (F-315) cells stained with the DAB reaction were examined under oil immersion. In the majority of the cells, mitochondria were recognized as discrete bodies in the thinner peripheral portion of the cytoplasm. This observation indicates that in a large proportion of HeLa F-315 cells, at least under the growth conditions used here, the mitochondrial complement is dividied into distinct organelles. This examination also revealed a considerable morphological heterogeneity of mitochondria, which exhibited an ovoid or short rod-like or, less frequently, long filamentous shape, with some evidence of branching. The variability in mitochondrial morphology appeared to be far more prounced between different cells than within individual cells; this cellular heterogeneity was not related in any obvious way to cell-cycle-dependent changes. 相似文献
17.
Synthesis of compound libraries and their concurrent assessment as selective reagents for probing and modulating biological function continues to be an active area of chemical biology. Microwave-assisted solid-phase Dötz benzannulation reactions have been used to inexpensively synthesize 2, 3-disubstituted-1, 4-naphthoquinone derivatives. Herein, we report the biological testing of a small library of such compounds using a murine fibroblast cell line (L929). Assessment of cellular viability identified three categories of cytotoxic compounds: no toxicity, low/intermediate toxicity and high toxicity. Increased levels of Annexin-V-positive staining and of caspase 3 activity confirmed that low, intermediate, and highly toxic compounds promote cell death. The compounds varied in their ability to induce mitochondrial depolarization and formation of reactive oxygen species (ROS). Both cytotoxic and non-cytotoxic compounds triggered mitochondrial depolarization, while one highly cytotoxic compound did not. In addition, all cytotoxic compounds promoted increased intracellular ROS but the cells were only partially protected from compound-induced apoptosis when in the presence of superoxide dismutase, catalase, or ascorbic acid suggesting utilization of additional pro-death mechanisms. In summary, nine of twelve (75%) 1, 4-naphthoquinone synthetic compounds were cytotoxic. Although the mitochondria did not appear to be a central target for induction of cell death, all of the cytotoxic compounds induced ROS formation. Thus, the data demonstrate that the synthesis regime effectively created cytotoxic compounds highlighting the potential use of the regime and its products for the identification of biologically relevant reagents. 相似文献
18.
Jasmonates are plant stress hormones that induce suppression of proliferation and death in cancer cells, while being selectively
inactive towards non-transformed cells. Jasmonates can overcome apoptotic blocks and exert cytotoxic effects on drug-resistant
cells expressing p53 mutations. Jasmonates induce a rapid depletion of ATP in cancer cells. Indeed, this steep drop occurs
when no signs of cell death are detectable yet. Experiments using modulators of ATP synthesis via glycolysis or oxidative
phosphorylation suggest that the latter is the pathway suppressed by jasmonates. Consequently, the direct effects of jasmonates
on mitochondria were evaluated. Jasmonates induced cytochrome c release and swelling in mitochondria isolated from cancer
cells but not from normal ones. Thus, the selectivity of jasmonates against cancer cells is rooted at the mitochondrial level,
and probably exploits differences between mitochondria from normal versus cancer cells. These findings position jasmonates
as promising anti-cancer drugs acting via energetic depletion in neoplastic cells. 相似文献
20.
The cytotoxicity of five Bacillus spp. isolated from alkaline cleaning solutions in South African dairies was evaluated against McCoy mouse cells using a 3-(4,5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT)-based assay, confocal scanning laser microscopy and scanning electron microscopy. According to the MTT-based assay, two of the Bacillus isolates (Bacillus licheniformis 5 and B. pumilus 122) were cytotoxic to McCoy cells and the cytotoxic components were heat labile. Confocal scanning laser microscopy combined with fluorescent staining using propidium iodide and fluorescein diacetate indicated that cytotoxic effects occurred within 3 h, appeared to be membrane active and resulted in cell necrosis. Scanning electron microscopy showed that McCoy cells exposed to the cytotoxic components exhibited morphological damage. 相似文献
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