carbocyanines and Carcinoma--Ehrlich-Tumor

Tumor development is the consequence of expanding the population of low differentiated cells with unlimited self-maintenance potential, i.e. cancer stem cells (CSCs). Application of new forms of nanocomposites capable of binding to CSCs and inducing the tumor destruction is perspective direction for treating this pathology. There have been developed the methods of obtaining hybrid nanocomplexes containing rare-earth orthovanadates GdYVO4:Eu³⁺, cholesterol and luminescent dye Dil. By immune fluorescence method using monoclonal antibodies to CD44, CD24, CD117 and Sca-1 markers there has been established the change in the ratio of tumor progenitors of various differentiation levels in a general pool of Ehrlich carcinoma (EC) after treatment with hybrid nanocomplexes. Essential reduction in the concentration of the most tumorogenic CD44high cells with simultaneous rise in the number of CD117⁺-cells resulted in an increased index of CD44high/CD117⁺ ratio. It has been demonstrated that application of hybrid nanocomplexes suppressed the tumor growth almost by 80%. The value of cooperative interactions of the cells with different phenotype signs in tumor sites has been proved. The index of CD44high/CD117⁺ ratio can be used as one of diagnostic and prognostic parameters of development and inactivation rate of tumor process when using different types of anti-tumor therapy.

Topics: Animals; Antibodies, Monoclonal; Antineoplastic Agents; Biomarkers, Tumor; Carbocyanines; Carcinoma, Ehrlich Tumor; Cholesterol; Europium; Female; Gadolinium; Gene Expression; Hyaluronan Receptors; Luminescence; Mice; Mice, Inbred BALB C; Nanocapsules; Neoplasm Transplantation; Neoplastic Stem Cells; Proto-Oncogene Proteins c-kit; Survival Analysis; Vanadates

Mitochondria are considered as the main source of reactive oxygen species (ROS) in the cell. For this reason, they have been recognized as a source of various pathological conditions as well as aging. Chronic increase in the rate of ROS production is responsible for the accumulation of ROS-associated damages in DNA, proteins, and lipids, and may result in progressive cell dysfunctions and, in a consequence, apoptosis, increasing the overall probability of an organism's pathological conditions. The superoxide anion is the main undesired by-product of mitochondrial oxidative phosphorylation. Its production is triggered by a leak of electrons from the mitochondrial respiratory chain and the reaction of these electrons with O(2). Superoxide dismutase (MnSOD, SOD2) from the mitochondrial matrix as well as superoxide dismutase (Cu/ZnSOD, SOD1) present in small amounts in the mitochondrial intramembrane space, convert superoxide anion to hydrogen peroxide, which can be then converted by catalase to harmless H(2)O. In this chapter, we describe a relation between mitochondrial membrane potential and the rate of ROS formation. We present different methods applicable for isolated mitochondria or intact cells. We also present experiments demonstrating that a magnitude and a direction (increase or decrease) of a change in mitochondrial ROS production depends on the metabolic state of this organelle.

Topics: Animals; Benzimidazoles; Brain; Calcium; Carbocyanines; Carcinoma, Ehrlich Tumor; Cell Fractionation; Cell Line, Tumor; Electron Transport; Fibroblasts; HeLa Cells; Humans; Hydrogen Peroxide; Membrane Potential, Mitochondrial; Mice; Microscopy, Confocal; Mitochondria; Oxygen Consumption; Phenazines; Reactive Oxygen Species; Superoxides

We prepared the liposome binding Sialyl Lewis X (SLX) on the surface in order to specifically and efficiently deliver substances (fluorescent materials, chemical substances, proteins, genes, etc.) to inflammation or tumor regions. The liposome with SLX (SLX-Lipo-Cy5.5), in which fluorescent substance Cy5.5 was included, was administered intravenously to arthritis or Ehrlich Ascites Tumor (EAT) bearing mouse, and the accumulation of liposome was observed using two types of in vivo fluorescent imaging equipment. The result was that the accumulation of SLX-Lipo-Cy5.5 to inflammation or tumor regions was significantly higher than the control liposome without sugar chain (Lipo-Cy5.5) at 24 and 48 h after administration. In addition, it was confirmed that this accumulation showed a shift of liposome from blood vessels to the surrounding tissues. Thus, it was proven that this liposome is useful not only as an in vivo bio-imaging reagent but also as a drug delivery system (DDS).

Topics: Animals; Arthritis, Experimental; Carbocyanines; Carcinoma, Ehrlich Tumor; Female; Fluorescence; Fluorescent Dyes; Inflammation; Lewis Blood Group Antigens; Liposomes; Mice; Mice, Inbred BALB C; Oligosaccharides; Sialyl Lewis X Antigen

The fluorescence intensity of the dye 1,1'-dipropylox-adicarbocyanine (DiOC3-(5] has been measured in suspensions of Ehrlich ascites tumor cells in an attempt to monitor their membrane potential (Vm) under different ionic conditions, after treatment with cation ionophores and after hypotonic cell swelling. Calibration is performed with gramicidin in Na+-free K-/choline-media, i.e., standard medium in which NaCl is replaced by KCl and cholineCl and where the sum of potassium and choline is kept constant at 155 mM. Calibration by the valinomycin "null point" procedure described by Laris et al. (Laris, P.C., Pershadsingh, A., Johnstone, R.M., 1976, Biochim, Biophys. Acta 436:475-488) is shown to be valid only in the presence of the Cl- -channel blocker indacrinone (MK196). Distribution of the lipophilic anion SCN- as an indirect estimation of the membrane potential is found not to be applicable for the fast changes in Vm reported in this paper. Incubation with DiOC3-(5) for 5 min is demonstrated to reduce the Cl permeability by 26 +/- 5% and the NO3- permeability by 15 +/- 2%, while no significant effect of the probe could be demonstrated on the K+ permeability. Values for Vm, corrected for the inhibitory effect of the dye on the anion conductance, are estimated at -61 +/- 1 mV in isotonic standard NaCl medium, -78 +/- 3 mV in isotonic Na+-free choline medium and -46 +/- 1 mV in isotonic NaNO3 medium. The cell membrane is depolarized by addition of the K+ channel inhibitor quinine and it is hyperpolarized when the cells are suspended in Na+-free choline medium, indicating that Vm is generated partly by potassium and partly by sodium diffusion. Ehrlich cells have previously been shown to be more permeable to nitrate than to chloride. Substituting NO3- for all cellular and extracellular Cl- leads to a depolarization of the membrane, demonstrating that Vm is also generated by the anions and that anions are above equilibrium. Taking the previously demonstrated single-file behavior of the K+ channels into consideration, the membrane conductances in Ehrlich cells are estimated at 10.4 microS/cm2 for K+, 3.0 microS/cm2 for Na+, 0.6 microS/cm2 for Cl- and 8.7 microS/cm2 for NO3-. Addition of the Ca2+-ionophore A23187 results in net loss of KCl and a hyperpolarization of the membrane, indicating that the K+ permeability exceeds the Cl- permeability also after the addition of A23187. The K+ and Cl- conductances in A23187-treated Ehrlich cells are estimated at 134 and

Topics: Animals; Calcimycin; Carbocyanines; Carcinoma, Ehrlich Tumor; Cell Membrane Permeability; Chlorides; Electric Conductivity; Fluorescent Dyes; Gramicidin; Ion Channels; Membrane Potentials; Mice; Quinine; Thiocyanates; Valinomycin

Topics: Animals; Carbocyanines; Carcinoma, Ehrlich Tumor; Cell Membrane; Membrane Potentials; Mice; Spectrometry, Fluorescence

It has been shown that in the absence of inhibitors of oxidative phosphorylation, changes in fluorescence of 3',3'-diethylthiodicarbocyanine iodide (dis-C2-/5/) in a cell suspension of Ehrlich's ascites carcinoma reflect those in the transmembrane mitochondrial potential. Addition of glucose to the cells in the presence of respiratory inhibitors similar to rotenon induces oscillations in the membrane mitochondrial potential due to H+-ATPase that uses glycolytic ATP. The described changes in energy metabolism parameters are determined by impairment of the interplay between glycolysis and oxidative phosphorylation. The low rate of cytoplasmic NADH oxidation by tumor cell mitochondria favors the latter's accumulation by the cytoplasm and glycolysis inhibition. Pyruvate has been shown to be responsible for NADH oxidation and to remove glycolysis inhibition.

Topics: Animals; Carbocyanines; Carbonyl Cyanide p-Trifluoromethoxyphenylhydrazone; Carcinoma, Ehrlich Tumor; Cells, Cultured; Energy Metabolism; Fluorescent Dyes; Glucose; Kinetics; Luminescent Measurements; Mice; Oligomycins; Oxygen Consumption; Pyrimidine Nucleotides; Quinolines; Rotenone

The addition of glucose to a suspension of Ehrlich ascites tumor cells results in rapid acidification of the extracellular medium due to lactic acid production. The nature of the H+ efflux mechanism has been studied by measuring the time course of the acidification, the rate of proton efflux, the direction and relative magnitude of the H+ concentration gradient, and the voltage across the membrane. Using the pH-sensitive dye acridine orange, we have established that after addition of 10 mM glucose an outward-directed H+ concentration gradient develops. As the rate of glycolysis slows, the continued extrusion of H+ reverses the direction of the H+ concentration gradient. Changes in absorbance of the voltage-sensitive dye diethyloxadicarbocyanine iodide (DOCC), and changes in the distribution of the lipid permeant cation tetraphenyl phosphonium, showed a dramatic and persistent hyperpolarization of the membrane voltage after glucose addition. The hyperpolarization was prevented by the protonophore tetrachlorosalicylanalide (TCS) and by valinomycin, but not by the neutral-exchange ionophore nigericin. Inhibitors of lactate efflux were found to reduce the rate of acidification after glucose addition but they had no effect on the magnitude of the resulting hyperpolarization. On the basis of these and other data we suggest that an active electrogenic pump mechanism for H+ efflux may be activated by glucose and that this mechanism operates independently of the lactate carrier system.

Topics: Acridine Orange; Animals; Carbocyanines; Carcinoma, Ehrlich Tumor; Cell Membrane; Coloring Agents; Glucose; Glycolysis; Hydrogen-Ion Concentration; Kinetics; Mice; Onium Compounds; Organophosphorus Compounds; Protons; Salicylanilides; Valinomycin

Uptake of alpha-aminoisobutyric acid (AIB) was examined in Ehrlich ascites tumor cells treated with the cation-exchange ionophore nigericin (20 microgram/ml). Membrane voltages were measured using the voltage-sensitive dye diethyloxadicarbocyanine (DOCC). In normal phosphate-buffered media, nigericin changed the distribution ratios of Na+ and K+ (the ratio of intra- to extracellular concentrations) nearly to unity, but AIB was still accumulated to a distribution ratio of approximately 9.0. When all but 40 mM Na+ in the medium was replaced by choline, nigericin resulted in K+ loss and Na+ gain and both cation distribution ratios approached 2.8-3.4, as would be expected if both ions were distributing near electrochemical equilibrium with a membrane voltage in the range of -28 to -33 mV. This conclusion was supported by the observation that the addition of 5 X 10(-7) M valinomycin to the nigericin-treated cell suspension produced no change in DOCC absorbance. In spite of the apparent zero electrochemical potential gradients for Na+ and K+, AIB was accumulated to a distribution ratio of 5.4 in the low-Na+ medium. Addition of 0.1 mM oubain or 50 microM vanadate did not alter the extent of AIB accumulation as would have been expected if a large component of the membrane voltage were due to electrogenic operation of the (Na+ + K+)-ATPase. Addition of lactate, pyruvate or glucose increased the AIB distribution ratios to 11.9, 9.4 and 15.3, respectively. The effect of glucose could be explained, at least in part, by an enhanced Na+ electrochemical potential gradient. However, neither lactate nor pyruvate produced any change either in membrane voltage or the intracellular Na+ concentration. Therefore, these results confirm the existence of a metabolic energy source which is coupled to AIB accumulation and operates in addition to the Na+ co-transport mechanism, and which is augmented by metabolic substrates such as lactate and pyruvate.

Topics: Amino Acids; Aminoisobutyric Acids; Animals; Biological Transport, Active; Carbocyanines; Carcinoma, Ehrlich Tumor; Coloring Agents; Kinetics; Membrane Potentials; Mice; Nigericin; Potassium; Sodium

The fluorescent dye, 3,3'-dipropylthiadicarbocyanine iodide, is frequently used to estimate the cell membrane potential of small cells. We have tested the effects of this dye on glycolytic rate, O2 consumption, and cellular ATP content in Ehrlich ascites tumor cells. Addition of the dye to steady state cells in the absence of glucose induces a rapid depletion (half-time approximately equal to 5 min) of cellular ATP, which is secondary to a 76% inhibition of O2 consumption. Aerobic glycolysis is stimulated by 76%. Valinomycin produces an additional stimulation of acid production. Our findings indicate that this fluorescent dye alters energy metabolism of Ehrlich ascites tumor cells. The response is consistent with primary inhibition of oxidative phosphorylation.

Topics: Adenosine Triphosphate; Animals; Benzothiazoles; Carbocyanines; Carcinoma, Ehrlich Tumor; Energy Metabolism; Glycolysis; Kinetics; Mice; Oxidative Phosphorylation; Oxygen Consumption; Quinolines; Valinomycin

Topics: Animals; Benzothiazoles; Biological Transport, Active; Carbocyanines; Carcinoma, Ehrlich Tumor; Cell Membrane; Fluorescent Dyes; Kinetics; Membrane Potentials; Mice; Ouabain; Potassium; Quinolines; Sodium

The cyanine dye 3,3'-dipropyl-2,2'-thiadicarbocyanine iodide [diS-C3(5)] was found to be a potent inhibitor of endogenous respiration of Ehrlich ascites tumor cells. This effect is believed to involve both rotenone and uncoupler type actions of the dye on the electron transport system of the mitochondria.

Topics: Adenosine Triphosphatases; Animals; Benzothiazoles; Carbocyanines; Carcinoma, Ehrlich Tumor; Dinitrophenols; Electron Transport; Fluorides; Kinetics; Mice; Mitochondria; Oligomycins; Oxygen Consumption; Quinolines; Rotenone

(1) (DL)-Propranolol and Ca2+ are shown to alter the transmembrane potential difference of Ehrlich ascites tumor cells as measured by means of the cyanine dye, 3,3'-dipropyl-2,2'-thiodicarbocyanine iodide, whose fluorescent intensity changes as a function of membrane potential. (2) The changes in membrane potential elicited by these agents are dependent of the external K+ concentration in a manner which suggest that the potential changes result from a specific increase in the permeability of the plasma membrane to K+. (3) Na+-dependent amino acid transport in the presence of propranolol can be modulated by varying the external K+ concentration (K+o). The initial rate of uptake is stimulated by propranolol at low K+o and inhibited at high K+o. The change in transport rate is nearly directly proportional to the natural logarithm of [K+]o in the presence of propranolol. (4) ATP depletion of the cells by preincubation with rotenone abolishes the changes in fluorescence and amino acid uptake seen with propranolol as a function of K+o. Restoration of cellular ATP with glucose in presence of Ca2+ restores both fluorescence and amino acid transport changes which occur in response to propranolol. (5) The fluorescence changes and amino acid transport changes in response to propranolol are pH dependent, with little effect seen at pH6. (6) It is concluded that the rate of Na+-dependent amino acid uptake is a function of membrane potential and is dependent on the electrochemical potential difference for Na+.

Topics: Adenosine Triphosphate; Amino Acids; Animals; Biological Transport, Active; Calcium; Carbocyanines; Carcinoma, Ehrlich Tumor; Hydrogen-Ion Concentration; Membrane Potentials; Ouabain; Potassium; Propranolol; Sodium; Valinomycin