Uptake of ferrous iron histidinate, a promoter of lipid peroxidation, by Ehrlich ascites tumor cells

The kinetics of the uptake of Fe(II)-histidinate, a known promoter of lipid peroxidation, into Ehrlich ascites tumor (EAT) cells and the intracellular binding of iron were studied in vitro. EAT cells (27.10(6)/ml) were incubated in Hanks' balanced salts solution at 37 degrees C for various time intervals in the presence of FeSO4 (1 mM) and L-histidine (10 mM). Total iron was determined by the 1,10-phenanthroline/ascorbate method and ferric iron by reaction with 5-sulfosalicylic acid; the difference was ascribed to ferrous iron. Total iron decreased rapidly in the medium (242 nmol within the first 10 min), and a corresponding increase of total iron (saturation value 376 nmol after 60 min) was determined within the cells, after the cellular proteins had been solubilized with 6 M urea. In the absence of EAT cells, Fe(II)-histidinate was readily oxidized to Fe(III)-histidinate by oxygen, but this reaction was strongly retarded by the tumor cells. The uptake of iron histidinate occurred in the oxidized state, while an uptake of ferrous iron could not be proven unambiguously. When EAT cells were saturated with iron, it was found that 93% of intracellular iron was bound to water-insoluble proteins and 7% was associated with soluble proteins, while no unbound iron was detectable by the method used. It was concluded that, despite the high uptake of total iron, only a very small portion of the intracellular iron was available as a redox catalyst for lipid peroxidation.     

Detection of 4-hydroxynonenal as a product of lipid peroxidation in native Ehrlich ascites tumor cells

4-Hydroxynonenal, which is a major product of lipid peroxidation in rat liver microsomes, was detected in native Ehrlich ascites tumor cells. Its formation was stimulated either by ferrous ions or by Fe(II)-histidinate. The identification was based on chromatographic (TLC/HPLC) and ultraviolet-spectroscopic evidence using synthetic 4-hydroxynonenal as reference. Highest values of 4-hydroxynonenal concentration (about 0.1 microM in the cell suspension) after 30 min of incubation were observed with Fe(II)-histidinate as stimulant. Saturation was already reached after an incubation period of 10 min. The results confirm the expectation by Schauenstein and Esterbauer (in Submolecular Biology and Cancer, Ciba Foundation Series 67 (1979) pp. 225-244, Excerpta Medica, Amsterdam) that endogenous lipid peroxidation gives rise to a distinct intracellular level of alpha, beta-unsaturated aldehydes. A simple hypothetical mechanism for the formation of 4-hydroxynonenal from n-6-polyunsaturated fatty acids is presented.     

Selective incorporation of various C-22 polyunsaturated fatty acids in Ehrlich ascites tumor cells

Three 14C-labeled 22-carbon polyunsaturated fatty acids, 7,10,13,16-[14C]docosatetraenoic acid (22:4(n-6)), 7,10,13,16,19-[14C]docosapentaenoic acid (22:5(n-3)), and 4,7,10,13,16,19-[14C]docosahexaenoic acid (22:6(n-3)), were compared with [3H]arachidonic acid (20:4(n-6] and [14C]linoleic acid (18:2(n-6)) to characterize their incorporation into the lipids of Ehrlich ascites cells. The relatively rapid incorporation of the labeled 22-carbon acids into phosphatidic acid indicated that substantial amounts of these acids may be incorporated through the de novo pathway of phospholipid synthesis. In marked contrast to 20:4(n-6), the 22-carbon acids were incorporated much less into choline glycerophospholipids (CGP) and inositol glycerophospholipids (IGP). No selective preference was apparent for the (n-3) or (n-6) type of fatty acids. The amounts of the acids incorporated into diacylglycerophosphoethanolamine were in the order of: 22:6(n-3) greater than 20:4(n-6) much greater than 22:5(n-3) greater than or equal to 22:4(n-6) greater than 18:2(n-6), whereas for alkylacylglycerophosphoethanolamine they were in the order of: 22:4(n-6) greater than 22:6(n-3) greater than 22:5(n-3) much greater than 20:4(n-6) greater than 18:2(n-6). Of the mechanisms possibly responsible for the selective entry of 22-carbon acids into ethanolamine glycerophospholipids, the most reasonable explanation was that the cytidine-mediated ethanolamine phosphotransferase may have a unique double selectivity: for hexaenoic species of diacylglycerol and for 22-carbon polyunsaturated fatty acid-containing species of alkylacylglycerol. The relative distribution of fatty acids between newly incorporated and already maintained lipid classes suggested that IGP may function in Ehrlich cells as an intermediate pool for the retention of polyunsaturated fatty acids in glycerolipids.     

Lipid peroxidation and cytotoxicity of Ehrlich ascites tumor cells by ferric nitrilotriacetate

Ferric nitrilotriacetate, which causes in vivo organ injury, induced lipid peroxidation and cell death in Ehrlich ascites tumor cells in vitro. The process was inhibited by butylated hydroxyanisole and enhanced by vitamin C and linolenic acid, indicating a close relationship between cytotoxicity and the lipid peroxidizing ability of Fe3+ NTA. The cytotoxicity was suppressed by glucose and a temperature below 20 degrees C. Lipid peroxidation of Fe3+ NTA-treated cells was greater at 0 degree C than at 37 degrees C, contrary to results with Fe3+ NTA-treated plasma membranes of Ehrlich ascites tumor cell. These results suggested that metabolism and membrane fluidity are important factors in the expression of the Fe3+ NTA-induced cytotoxicity. H2O2 showed a lower cytotoxicity than did Fe3+ NTA but a greater lipid peroxidizing ability. H2O2 appeared to damage the cells less, and was quenched rapidly by cellular metabolism unlike Fe3+ NTA. In transferrin-free medium, Ehrlich ascites tumor cell readily incorporated Fe3+ NTA, and iron uptake was greater than NTA-uptake in Fe3+ NTA-treated cells, suggesting that Ehrlich ascites tumor cell incorporated iron from Fe3+NTA and metabolized it into an inert form such as ferritin.     

声化学激活血卟啉诱导艾氏腹水肿瘤细胞凋亡

本实验采用频率为2．0MHz，声强分别为1．0w／cm^2、1．5w／cm^2、2．0w／cm^2等不同参数，研究超声激活血卟啉对艾氏腹水肿瘤细胞的杀伤作用和诱导肿瘤细胞凋亡现象。通过扫描电镜、透射电镜以及荧光显微镜观察受损后细胞形态结构的变化，主要表现为细胞微绒毛的减少，胞膜结构和通透性的改变，细胞器的受损以及核物质的分解、丢失；同时发现处理后的肿瘤细胞有核物质凝集、趋边排列以及凋亡小体的形成等细胞凋亡特征。研究中首次发现声化学激活血卟啉在对艾氏腹水肿瘤细胞杀伤的同时，也能诱导艾氏腹水肿瘤细胞发生凋亡，提示在声动力疗法中并存着对癌细胞的直接杀伤和通过诱导癌细胞凋亡的两种抗癌途径。     

Selective acylation of alkyllysophospholipids by docosahexaenoic acid in Ehrlich ascites cells

Ehrlich ascites cells were cultured with 1-O-[3H]alkylglycero-3-phosphoethanolamine (1-[3H]alkyl-GPE) or 1-O-[3H]alkylglycero-3-phosphocholine (1-[3H]alkyl-GPC) to reveal the selective retention of polyunsaturated fatty acids at second position of ether-containing phospholipids. Although small percentages of the lysophospholipids were degraded into long-chain alcohol, both alkyllyso-GPE and -GPC were acylated at the rate of approximately 2 nmol/30 min per 10(7) cells. Alkylacylacetylglycerols were prepared from the acylated products by phospholipase C treatment, acetylation and TLC, and fractionated according to the degree of unsaturation by AgNO3-TLC. The distribution of the radioactivity among the subfractions indicated that both alkyllysophospholipids were mainly esterified by docosahexaenoic acid and to a somewhat lesser extent by arachidonic acid. The selectivity for docosahexaenoic acid in the esterification of 1-alkyl-GPE was much stronger than in that of 1-alkyl-GPC. Although acyl-CoA: 1-alkyl-glycerophosphoethanolamine acyltransferase activity of Ehrlich cell microsomes with arachidonoyl-CoA and docosahexaenoyl-CoA as acyl donors was negligible compared with the acyl-CoA:1-alkyl-glycerophosphocholine acyltransferase activity, a significant amount of 1-alkyl-GPE was acylated in the microsomes without exogenously added acyl-CoA. HPLC analysis revealed that docosahexaenoic acid and arachidonic acid were mainly esterified by the microsomal transferase. Acylation of 1-alkyl-GPC with docosahexaenoic acid and arachidonic acid was also observed in the absence of added acyl-CoA, but the activity was lower than that for 1-alkyl-GPE. Although the source of the acyl donor in the acylation has not been determined, the acylation is probably due to the direct transfer of acyl groups between intact phospholipids. The above results provided the first evidence that the lysophospholipid acyltransferase system including the transacylase activity participates in the selective retention of docosahexaenoic acid in intact cells and a cell free system.     

Permeabilization of Ehrlich ascites tumor cells by human serum

Human serum rapidly permeabilized Ehrlich ascites tumor cells to inorganic cations such as Rb⁺ and Ca²⁺; serum from several other species showed little or no activity. The effect of human serum was not reversed by washing the cells. Human serum, deficient in specific complement proteins, had no activity, but was reactivated by the addition of the missing complement component. Since Ca²⁺ was not required for the permeabilization, the alternative pathway of complement activation was implicated. Human serum deficient in Factor B of the alternative pathway was ineffective, but permeabilizing activity was restored by addition of Factor B. Rb⁺ uptake of several other cells was not inhibited by human serum. We conclude that an interaction between human complement and Ehrlich ascites tumor cells is responsible for the membrane lesion observed.     

Role of protein dissociation in the transport of acidic amino acids by the Ehrlich ascites tumor cell.

The pH profile for the uptake of L-glutamic acid by the Ehrlich ascites tumor cell arises largely as a sum of the decline with falling pH of a slow, Na+-dependent uptake by System A, and an increasing uptake by Na+-independent System L. The latter maximizes at about pH 4.5, following approximately the titration curve of the distal carboxyl group. This shift in route of uptake was verified by (a) a declining Na+-dependent component, (b) an almost corresponding decline in the 2-(methylamino)-isobutyric acid-inhibitable component, (c) a rising component inhibited by 2-aminonorbornane-2-carboxylic acid. Other amino acids recognized as principally reactive with Systems A or L yielded corresponding inhibitory effects with some conspicious exceptions: 2-Aminoisobutyric acid and even glycine become better substrates of System L as the pH is lowered; hence their inhibitory action on glutamic acid uptake is not lost. The above results were characterized by generally consistent relations among the half-saturation concentrations of the interacting amino acids with respect to: their own uptake, their inhibition of the uptake, one by another, and their trans stimulation of exodus, one by another. A small Na+-dependent component of uptake retained by L-glutamic acid but not by D-glutamic acid at pH 4.5 is inhibitable by methionine but by neither 2-(methylamino)-isobutyric acid nor the norbornane amino acid. We provisionally identified this component with System ASC, which transports L-glutamine throughout the pH range studied. No transport activity specific to the anionic amino acids was detected, and the unequivocally anionic cysteic acid showed neither significant mediated uptake nor inhibition of the uptake of glutamic aic or of the norbornane amino acid. The dicarboxylic amino acids take the sequence, aspartic acid less than glutamic acid less than alpha-aminoadipic acid less than S-carboxymethylcysteine, in their rate of mediated, Na+-independent uptake at low pH. Diiodotyrosine and two dissimilas isomers of nitrotyrosine also show acceleration of uptake as the phenolate group on the sidechain is protonated, a result indicating that the acidic group need not be a carboxyl group and need not take a specific position in space to be accepted at the receptor site L. The presence of the carboxyl group does not upset the normal stereospecificity of System L until it falls on the beta-carbon in aspartic acid; even then it is the presence of the carbonyl group and not of the intact carboxyl group nor of its hydroxyl group that cancels out the stereospecificity, as was shown by the absence of normal stereospecificity for aspartic acid and asparagine and its presence in glutamic acid, homoserine and glutamine. In agreement, the uptak of aspartic acid is peculiarly sensitive to the presence of an alpha-methyl group or of other structures that modify the orientation of the sidechain.     

Reconstitution of neutral amino acid transport system from Ehrlich ascites tumor cells.

Amino acid transport systems for alanine and leucine have been reconstituted into artificial lipid vesicles. Purified plasma membrane vesicles from Ehrlich ascites cells were dissolved in 2% sodium cholate, 1 mM dithiothreitol, 0.5 mM EDTA, a mixture which solubilized approximately 50% of the membrane protein. This solubilized protein fraction was further purified by a combination of ammonium sulfate precipitations, gel filtration, and DEAE-cellulose chromatography. A fraction containing approximately 15 Coomassie blue staining bands on sodium dodecyl sulfate gels was obtained. This material was reconstituted into liposomes, and preliminary results demonstrated transport of alanine and leucine dependent on a sodium gradient. In addition, an electrogenic gradient mediated by valinomycin-induced potassium diffusion seemed to stimulate alanine uptake further.     

Changes in adenylate energy charge in Ehrlich ascites tumor cells deprived of serum, glucose, or amino acids.

The adenylate energy charge in Ehrlich ascites tumor cells increases when cells are cultivated in serum-limiting medium and decreases when they are incubated in glucose- or amino acid-limited media. Protein synthetic rates decrease in cells deprived of serum, glucose, or amino acids. Supplementation of deprived cells with respective nutrients restores normal protein synthetic rates and adenylate energy charge values. Serum-deprived cells incubated in depleted serum media do not increase their rates of protein synthesis and their adenylate energy charge remains elevated. These results suggest that serum factors regulate protein synthetic rates by mechanisms other than those regulating the availability in cells of glucose or of amino acids. The increased rates of utilization of glucose and of amino acids following the addition of serum are probably due to increased biosynthetic requirements.     

Membrane fluidity in Ehrlich ascites tumor cells treated with adriamycin

The incubation of Ehrlich ascites tumor cells with adriamycin resulted in an increase in lipid peroxide content and a decrease in membrane fluidity as measured by electron spin resonance using the paramagnetic probe 5-doxylstearic acid. Coincidently, the incorporation of [3H]thymidine into tumor cells was progressively inhibited as the concentration of adriamycin was increased. The results indicate that adriamycin induces changes in the plasma membrane of Ehrlich ascites tumor cells after exposure to a low, but cytotoxic, level of this agent.