Inhibition of cell proliferation by putative metabolites and non-degradable analogs of methotrexate-gamma-dimyristoylphosphatidylethanolamine

Previous investigations have shown that untargeted liposomes, in which methotrexate is anchored to the lipid bilayers as methotrexate-gamma-dimyristoylphosphatidylethanolamine (methotrexate-gamma-DMPE), can inhibit in vitro cell proliferation. To test the possibility that this inhibition may involve extracellular metabolism of methotrexate-gamma-DMPE, we have degraded it chemically (dilute alkali) or enzymatically (phospholipase A2, phospholipase C, phospholipase C plus phosphatase), and assayed the products using human lymphoblastoid T cells or a subline that has a defective methotrexate transport system. Neither methotrexate-gamma-(1-myristoyl)-glycerophosphorylethanolamine, methotrexate-gamma-glycerophosphorylethanolamine, methotrexate-gamma-phosphorylethanolamine, nor methotrexate-gamma-ethanolamine resemble methotrexate-gamma-DMPE sensitized liposomes or the free derivative in their ability to block tritiated deoxyuridine incorporation into DNA. When added extracellularly, these putative metabolites manifest a higher ID50 concentration and/or, unlike the liposomes or unincorporated methotrexate-gamma-DMPE, utilize the methotrexate transport system to enter cells. Additionally, we have synthesized methotrexate-gamma-dihexadecylphosphatidylethanolamine and methotrexate-gamma-hexadecylphosphorylethanolamine, analogs of methotrexate-gamma-DMPE that cannot be hydrolyzed by phospholipases A2, C and D; liposomes prepared with these derivatives are markedly less potent cytotoxic agents than methotrexate-gamma-DMPE sensitized liposomes. All together, these results are consistent with the conclusion that methotrexate-gamma-DMPE must undergo intracellular metabolism to exert optimal inhibition; they also bear on possible mechanisms by which methotrexate-gamma-DMPE may enter cells.     

Liposome-mediated delivery of pteridine antifolates to cells in vitro: potency of methotrexate,and its α and γ substituents

We have examined the growth-inhibitory potency of several pteridines encapsulated in negatively charged liposomes, including methotrexate, methotrexate-γ-methylamide, methothrexate-γ-dimethylamide, methotrexate-α-aspartate, and a lipophilic methotrexate-phosphatidylethanolamine conjugate. The potency of encapsulated methotrexate is greater than the potency of the free drug for CV1-P cells, but not for other cell lines. The potency of methotrexate-γ-methylamide and mehtotrexate-γ-dimethylamide is only minimally improved by encapsulation. The potency of methotrexate-α-aspartate is increased by encapsulation. In addition, the lipophilic methotrexate derivative has demonstrable potency when incorporated in liposomes. We have also examined the potency of several pteridines under conditions where cells are exposed to the drug for periods shorter than the entire growth assay. Reduction of the exposure time decreases the potency of both encapsulated and free drugs. However, the difference in potency between the encapsulated and free drug is increased, because the potency of the encapsulated drug is affected less. Consequently, encapsulated methotrexate-γ-aspartate is 300-fold more potent than free drug, if CV1-P cells are exposed to drug for 4 h. Moreover, encapsulated methotrexate is more potent than free methotrexate for growth inhibition of L929 fibroblasts, if the term of exposure is less than 8 h. Potency is least affected by reduction of exposure length for the lipophilic methotrexate derivative.     

Inhibition of cell proliferation with antibody-targeted liposomes containing methotrexate-γ-dimyristoylphosphatidylethanolamine

We have prepared liposomes containing methotrexate-γ-dimyristoylphosphatidylethanolamine (MTX-DMPE liposomes), to which protein A was covalently coupled, permitting specific association of these liposomes in vitro with murine cells preincubated with relevant protein A-binding monoclonal antibodies. In the absence of antibody the presence of externally-oriented methotrexate (MTX) in MTX-DMPE liposomes did not result in greater binding to cells than liposomes made without MTX-γ-DMPE. Derivation of methotrexate with phospholipid permits enhanced drug-liposome association. These liposomes are more resistant than conventional liposomes to repeated cycles of freezing and thawing. MTX-DMPE liposomes are comparable to antibody-targeted liposomes made with encapsulated water-soluble methotrexate both with respect to specific binding to target cells and drug effect. The inhibitory effects off MTX-liposomes, as well as free MTX, were reversible by either thiamin pyrophosphate (Tpp) or N⁵-formyltetrahydrofolate (F-THF), while the effects of MTX-DMPE liposomes were reversed only by N⁵-formyltetrahydrofolate. This suggests that the toxicity of non-targeted MTX-liposomes may be due to leakage of the encapsulated MTX. The absence of an effect of thiamin pyrophosphate on non-targeted MTX-DMPE liposomes indicates that they do not enter into the cell via the normal folate transport system.     

In vitro drug delivery mediated by ecto-NAD+-glycohydrolase ligand-targeted liposomes

We have studied the growth-inhibitory potency of methotrexate and methotrexate-γ-aspartate encapsulated in liposomes conjugated to ligands of ecto-NAD⁺-glycohydrolase (Salord, J. et al., Biochim. Biophys. Acta 886 (1986) 64–75). The ability of targeted liposomes to enhance growth inhibition, which amounted to a 4-fold reduction of the drug concentration required to inhibit cell growth by 50% as compared to nontargeted liposomes, was observed only with cells expressing this ecto-enzyme activity, i.e., Swiss 3T3 fibroblasts and RAJI, a Burkitt-type lymphoma cell line. Delivery of the encapsulated drugs was inhibited by NH₄Cl and varied with the endocytic capacity of the cells. Only small unilamellar vesicles affected the growth of the lymphoma cells, whereas the fibroblasts were more sensitive to large unilamellar vesicles. With vesicles of appropriate size, there was a good correlation between the specific binding of the targeted liposomes to cells and drug delivery. Our results suggest that ecto-NAD⁺-glycohydrolase can provide a recognition site on target cells and mediate the internalization of targeted liposomes by a mechanism most probably related to adsorptive endocytosis.     

Further studies on the charge-related alterations of methotrexate transport in Ehrlich ascites tumor cells by ionic liposomes: Correlation with liposome-cell association

Summary Interaction of positively (phosphatidylcholine/stearylamine 51) or negatively (phosphatidylcholine/stearic acid 51) charged liposomes with Ehrlich ascites tumor cells for 1–5 min increases or decreases, respectively, the bidirectional fluxes of the folic acid analog, methotrexate. These effects on influx and efflux appear to be symmetrical since the liposomes do not change the intracellular level of methotrexate at the steady state. Influx kinetics show that these alterations result from an increase or decrease in theV _max with no change in theK _m ⁱⁿ . These effects appear to be specific for the methotrexate-tetrahydrofolate carrier system since the transport of other compounds which utilize this carrier, aminopterin, 5-methyltetrahydrofolate, and 5-formyltetrahydrofolate, is affected similarly to methotrexate, whereas, the transport of folic acid, a compound similar in structure and charge but not significantly transported by this carrier is unaffected by liposomes. Once cells are exposed to charged liposomes, the effects on methotrexate transport cannot be reversed by washing the cells free of the extracellular liposomes. If, however, cells are exposed to liposomes of one charge, washed and then exposed to liposomes of the opposite charge, methotrexate influx is reversed to control rates. The effects of charged liposomes on methotrexate influx were not abolished by treating the cells with neuraminidase, metabolic inhibitors or lowering the temperature to 4°C. Studies on the uptake of [¹⁴C] liposomes show that these effects are not proportional to the total amount of lipid associated with the cell but result from an initial rapid liposome-cell association that is not dependent on temperature or energy metabolism nor related to cell surface charge.     

Inhibition of cell proliferation and dihydrofolate reductase by liposomes containing methotrexate-dimyristoylphosphatidylethanolamine derivatives and by the glycerophosphorylethanolamine analogs

Liposomes, which were prepared with the three methotrexate (MTX)-dimyristoylphosphatidylethanolamine (DMPE) derivatives described in the preceding paper, were tested for their ability to block proliferation of mouse 3T3 and L1210 cells. Tritiated deoxyuridine incorporation into DNA could be completely inhibited by liposomes sensitized with MTX-DMPE I (MTX-gamma-DMPE). Under similar conditions, liposomes containing MTX-DMPE II (MTX-alpha-DMPE) and MTX-DMPE III (MTX-alpha, gamma-diDMPE) produced partial and no inhibition, respectively. These effects on cell growth were paralleled by the capacity of liposomes, prepared with each of the DMPE derivatives, to inhibit dihydrofolate reductase isolated from L1210 cells. Analogous experiments with the three corresponding glycerophosphorylethanolamine (glyceroPE) analogs also indicated that MTX-glyceroPE I was the most effective inhibitor of both cell proliferation and enzymatic activity. However, MTX-DMPE I sensitized liposomes apparently enter target cells as a consequence of phagocytosis, and not via the ubiquitous methotrexate transport system that is employed by MTX-glyceroPE I. For example, novel use of thiamine pyrophosphate showed that this compound had no influence on inhibition of cell proliferation due to liposomes, whereas thiamine pyrophosphate could completely antagonize the inhibitory effects of methotrexate and MTX-glyceroPE I. The results are discussed with reference to possible therapeutic advantages of these liposomes.     

The effects of liposome size and surface charge on liposome-mediated delivery of methotrexate-γ-aspartate to cells in vitro

We have studied the liposome-mediated delivery of methotrexate-γ-aspartate to five cell lines. The sensitivity of the cells to encapsulated drug varies widely in accordance with their ability to take up the liposomes. CV1-P cells can be 150-times more sensitive to encapsulated methotrexate-γ-aspartate than to free drug, while AKR/J SL2 cells are only twice as sensitive to the encapsulated drug. Negatively-charged liposomes are much more efficient for delivery than are neutral liposomes, and cholesterol is an essential component of the liposome membrane for optimal drug delivery. The optimal liposome size for drug delivery is 0.1 μm, although the amount of cell-associated lipid is the same for all liposome sizes. The effect of the encapsulated drug is inhibited by NH₄Cl, suggesting an endocytic mechanism for delivery. The potency of the encapsulated drug is not affected by wide variations in the drug:lipid ratio.     

Alterations of the carrier-mediated transport of an anionic solute,methotrexate, by charged liposomes in Ehrlich ascites tumor cells

Summary Interaction of positively charged liposomes with Ehrlich ascites tumor cells increases the bidirectional transmembrane fluxes of the anionic folic acid analog, methotrexate. Negative liposomes reduce methotrexate influx. Stimulation of methotrexate influx by positively charged liposomes is time and concentration dependent, requiring at least a 5-min incubation with 2.5mm phosphatidylcholine containing 20% stearylamine for maximum effect. Stimulation is not appreciably reversed by washing the cells. Similar increases are observed for influx and efflux so that there is no change in the steady-state methotrexate electrochemical-potential difference across the cell membrane. The increase in influx appears to be a stimulation of the carrier-mediated transport process for methotrexate since both control and stimulated influx are abolished by the competitive inhibitor, 5-formyltetrahydrofolate or the sulfhydryl group inhibitor,p-chloromercuriphenylsulfonic acid and the Q₁₀ of the system remains unchanged. Influx of 5-methyltetrahydrofolate, which shares the same transport carrier as methotrexate, is also stimulated. However, the transport of folic acid, which is structurally similar to methotrexate but does not utilize the carrier, is unaffected. The kinetic change induced by positively charged liposomes is an increase in theV _ma ⁱⁿ , while theK _t ⁱⁿ remains unchanged. Trans-stimulation of methotrexate influx by 5-formyltetrahydrofolate occurs to the same extent in the presence or absence of positively charged liposomes. The liposomes have no apparent effect on the intracellular water, the extracellular space, or the chloride distribution ratio. The data suggest that interaction of positively charged liposomes with Ehrlich ascites tumor cells accelerates the rate of transposition of the membrane carrier system for methotrexate, altering the kinetics of transport without a change in transport thermodynamics.     

Effect of liposomally encapsulated MTX-DMPE conjugates upon TNFα and PGE2 release by lipopolysaccharide stimulated rat peritoneal macrophages

The ability of liposomally encapsulated preprations of methotrexate (MTX) and three of its lipophilic derivatives (MTX-γ-DMPE, MTX-α-DMPE and MTX-α,γ-diDMPE) to alter mediator release by lipopolysaccharide (LPS)-stimulated rat peritoneal macrophages (PMΘ) was investigated. The viability of these macrophages when incubated with approximately 6.0 nmol/10⁵ cells of the respective liposomal preparations (MTX-LIPO, MTX-γ-LIPO, MTX-α-LIPO and MTX-di-LIPO) for 20 h was greater than 80%. Treatment of macrophages, which had been incubated with MTX-α-LIPO (5.5 nmol/10⁵ cells), MTX-γ-LIPO (6.9 nmol/10⁵ ± 9.6%, 80.6 ± 5.6% and 91 ± 11.4% phagocytosis respectively (mean ± S.E.M.). At similar concentratio MTX-α-LIPO MTX-γ-LIPO and MTX-di-LIPO (6.5 nmol/10⁵ cells), PGE₂ release from LPS-stimulated rat peritoneal macrophages was inhibited by 85.3% ± 3.7%, 68.7 ± 0.6% and 88.8 ± 2.2% respectively (mean ± S.E.M., n = 4). Incubation of these macrophages with 12, 10 and 9.4 nmol/10⁵ cells of the respective liposomal preparations resulted in 89 ± 3.3%, 62 ± 5.5% and 85 ± 3.9% inhibition of TNF_α release (rmmean ± S.E.M., n = 4). However, at this concentration MTX-di-LIPO was toxic. Neither MTX (20?2.5 nmol/10⁵ cells) nor MTX-LIPO (5.6 nmol/10⁵ cells) affected TNF_α release from LPS-stimulated macrophages. Whilst free MTX wasl also ineffective at inhibiting PGE₂ from these cells, incubation with MTX-LIPO at the above concentration resulted in 76.9 ± 2.6% inhibition of the prostaglandins release.     

Association of Methotrexate Encapsulated in Negatively Charged Liposomes with Cells at Nanomolar Lipid Concentrations

Abstract

An in vitro liposome-cell association system has been developed that will allow the study of uptake and metabolism of liposomes by cultured cells at nanomolar lipid concentrations. The fate of cell associated liposomes is followed through the liposome encapsulated marker, methotrexate. Detection is based on the inhibition of dihydrofolate reductase by methotrexate, after its release from cells through boiling. Methotrexate in phospha-tidylglycerol (PG) liposomes is taken up by cells and then subsequently lost from the cells. Uptake is partially blocked by monensin. Loss from the cells is blocked by metabolic inhibitors, monensin, ammonium chloride, and chloroquine. Methotrexate in distearoylphosphatidylglycerol (DSPG) liposomes is taken up by cells slowly, and there is minimal lost of methotrexate after uptake. Pulse studies show that metabolism of PG liposomes after endocytosis is occurring at a much higher rate than that of DSPG liposomes, and substantial retention of encapsulated methotrexate occurs for both liposome compositions.     

Phospholipases: melittin facilitation of bee venom phospholipase A2-catalyzed hydrolysis of unsonicated lecithin liposomes.

Melittin isolated from the venom of the common honey bee is a potent activator for bee venom phospholipase A₂-catalyzed hydrolysis of unsonicated liposomes of egg phosphatidyl choline. At 37 °C and pH 8, the rate of this enzymatic reaction is increased approximately 300-fold by the addition of 8 × 10^?5m melittin. The magnitude of facilitation of the phospholipase A₂ reaction is much greater than that previously reported by other workers for systems involving sonicated egg phosphatidyl choline liposomes or Escherichia coli membrane fragments as substrates. Melittin having lysines quantitatively modified through reaction with methyl acetimidate is as effective a potentiator of phospholipase A₂ activity as the unmodified material. The same result was obtained for melittin in which the single tryptophan residue was modified. Melittin modified by succinylation retained approximately 50% of its capacity to facilitate phospholipase A₂ activity. In contrast, a modified melittin in which the C-terminal four amino residues were removed, acetimidated des(23–26)melittin, is a very poor activator, as is a mixture of this peptide with the C-terminal tetrapeptide. In contrast to the results with egg lecithin liposomes, melittin has little influence on the susceptibility of monomolecular aqueous solutions of dihexanoylphosphatidyl choline to phospholipase A₂ attack.     

Effect of trypsin, phospholipases, and membrane-impermeable reagents on the uptake of palmitic acid by isolated rat liver cells

Rat liver cells isolated by the collagenase-hyaluronidase perfusion method were treated with membrane-impermeable protein reagents (7-diazonium, 1–3-naphthalene disulfonate, diazotized sulfanilic acid, 8-anilino-naphthalene disulfonate), trypsin, phospholipase A, phospholipase C, and phospholipase D. The treated cells were incubated with [1-¹⁴C]palmitate and the ¹⁴CO₂ produced was taken as a measure of fatty acid uptake by the cells. ¹⁴CO₂ production by the cells was not inhibited after treatments with the membrane-impermeable protein reagents or phospholipase D. Treatments with small amounts of trypsin or phospholipases A or C caused inhibition of CO₂ production from tracer amounts of palmitate. The inhibition by trypsin was partially, and that by phospholipase A was fully, reversed by increasing the amount of palmitic acid in the incubation medium. The oxidation of shorter-chain fatty acids such as octanoic acid was not decreased but increased after treating the cells with trypsin or phospholipase A. The membrane-impermeable reagents inhibited the oxidation of palmitate to CO₂ by liver cells isolated by mechanical dispersion. These reagents also inhibited the long-chain acyl CoA ligase activity of liver microsomes. From these results it is suggested that the inhibition of CO₂ production by intact liver cells from palmitate after enzyme treatments, is due to partial removal or modification of a normal transport component for long-chain fatty acids on the plasma membrane. The possibility of proteins (or lipoproteins) buried below the surface layer of plasma membrane in fatty acid uptake by liver cells is indicated.     

Effect of purified phospholipases on the binding of tetrodotoxin to axon plasma membrane

Summary The role of phospholipids in the binding of [³H] tetrodotoxin to garfish olfactory nerve axon plasma membrane was studied by the use of purified phospholipases. Treatment of the membranes with low concentrations of either phospholipase A₂ (Crotalus adamanteus andNaja naja) or phospholipase C (Bacillus cereus andClostridium perfringens) resulted in a marked reduction in tetrodotoxin binding activity. A 90% reduction in the activity occurred with about 45% hydrolysis of membrane phospholipids by phospholipase A₂, and with phospholipase C the lipid hydrolysis was about 60–70% for a 70–80% reduction in the binding activity. Phospholipase C fromB. cereus andCl. perfringens had similar inhibitory effects. Bovine serum albumin protected the tetrodotoxin binding activity of the membrane from the inhibitory effect of phospholipase A₂ but not from that of phospholipase C. In the presence of albumin about 25% of the membrane phospholipids remained unhydrolyzed by phospholipase A₂. It is suggested that these unhydrolyzed phospholipids are in a physical state different from the rest of the membrane phospholipids and that these include the phospholipids which are directly related to the tetrodotoxin binding component. It is concluded that phospholipids form an integral part of the tetrodotoxin binding component of the axon membrane and that the phospholipase-caused inhibition of the binding activity is due to effects resulting from alteration of the phospholipid components.     

Intrinsic perturbing ability of alkanols in lipid bilayers

The proportionality constant between the equipotency concentrations of a series of solutes and the fraction of a solute in the membrane phase is directly related to the solute to lipid mol ratio. Experimental measurements of partition coefficient and of several alkanol-induced effects show that the solute/lipid mol ratlos for a series of alkanols are not constant at their equipotency concentrations. The deviations in the solute/lipid ratios are similar in the various systems, and these deviations seem to depend primarily upon the chain length and branching in alkanols. It is suggested that such intrinsic differences in the perturbing ability of alcohols arise from a specificity of interaction between alkanols and lipid bilayer. We have correlated partition coefficients (in n-octanol, in egg phosphatidylcholine liposomes, and in dipalmitoyl phosphatidylcholine liposomes) for thirteen alkanols to the equipotency concentrations for their ability to modify the order-disorder thermotropic transition in dipalmitoyl phosphatidylcholine, ability to stimulate the hydrolysis of phosphatidylcholine in a bilayer by bee venom phospholipase A₂, and for the activation of the galactoside transport system in Escherichia coli. Significant correlation is found between equipotency concentrations for perturbing the order-disorder transition, the activation of phospholipase A₂-catalyzed hydrolysis and the activation of galactoside transport system.     

Action of phospholipase A2 and phospholipase C on Escherichia coli

The action of exogenous phospholipases on Escherichia coli has been examined. Cells harvested in late log phase were found to be completely resistant to the action of phospholipases A₂ and C. Treatment of cells with Tris and EDTA was required to make the phospholipids in the cell accessible to these phospholipases. Phospholipase A₂ hydrolyzed mainly phosphatidylethanolamine and phosphatidylglycerol, whereas phospholipase C preferentially degraded phosphatidylethanolamine.During the EDTA treatment, an endogenous phospholipase A₁ or a lysophospholipase (or both) was unmasked which caused the formation of free fatty acids in experiments in which no phospholipase was added and which degraded some of the lysophospholipids formed by phospholipase A₂.The cells were rapidly killed by the successive Tris-EDTA-phospholipase treatment, but no cell disintegration was observed.     

Use of phospholipase A to compare phospholipid organization in synaptic membranes,myelin, and liposomes

Summary The pattern of fatty acid release from rat synaptic membranes in the presence of phospholipase A₂ (Vipera russelli) was compared to that from liposomes comprised of phospholipids. Phospholipase A₂ more readily attacked myelin and synaptic membranes than liposomes prepared from total phospholipids derived from myelin. Although hydrolysis of liposomal phospholipids occurred in the absence of added calcium, the presence of 2mm CaCl₂ or 2% bovine serum albumin significantly enhanced the phospholipase attack of liposomes, but not synaptic membranes or myelin. Phospholipase exhibited a marked preference for phospholipids containing docosahexaenoic acid (226) in the synaptic membranes, while with liposomes the pattern of released fatty acid reflected the fatty acid composition in the two-position of the phospholipids. Although either calcium or albumin markedly increased the phospholipase hydrolysis of liposomes, neither affected the hydrolysis of synaptic membranes or the pattern of fatty acid release from liposomes. It was concluded that the nonlipid constituents, particularly the proteins, of biomembranes were responsible for the organization of the phospholipids and accounted for the observed differences between liposomes and synaptic membranes with respect to enzymic accessibility.     

Arachidonic acid is an autocoid mediator of the differential action of 1,25-(OH)2D3 and 24,25-(OH)2D3 on growth plate chondrocytes

Prior studies have shown that 24,25-(OH)₂D₃ and 1,25-(OH)₂D₃ regulate protein kinase C (PKC) in costochondral chondrocytes in a cell maturation-dependent manner, with 1,25-(OH)₂D₃ affecting primarily growth zone (GC) cells and 24,25-(OH)₂D₃ affecting primarily resting zone (RC) cells. In addition, 1,25-(OH)₂D₃ has been shown to increase phospholipase A₂ activity in GC, while 24,25-(OH)₂D₃ has been shown to decrease phospholipase A₂ activity in RC. Stimulation of phospholipase A₂ in GC caused an increase in PKC, whereas inhibition of phospholipase A₂ activity in RC cultures increased both basal and 24,25-(OH)₂D₃-induced PKC activity, suggesting that phospholipase A₂ may play a central role in mediating the effects of the vitamin D metabolites on PKC. To test this hypothesis, RC and GC cells were cultured in the presence and absence of phospholipase A₂ inhibitors (quinacrine and oleyloxyethylphosphorylcholine [OEPC]), phospholipase A₂ activators (melittin and mastoparan), or arachidonic acid alone or in the presence of the target cell-specific vitamin D metabolite. PKC specific activity in the cell layer was determined as a function of time. Phospholipase A₂ inhibitors decreased both basal and 1,25-(OH)₂D₃-induced PKC activity in GC. When phospholipase A₂ activity was activated by inclusion of melittin or mastoparan in the cultures, basal PKC activity in RC was reduced, while that in GC was increased. Similarly, melittin and mastoparan decreased 24,25-(OH)₂D₃-induced PKC activity in RC and increased 1,25-(OH)₂D₃-induced PKC activity in GC. For both cell types, the addition of arachidonic acid to the culture media produced an effect on PKC activity that was similar to that observed when phospholipase A₂ activators were added to the cells. These results demonstrate that vitamin D metabolite-induced changes in phospholipase A₂ activity are directly related to changes in PKC activity. Similarly, exogenous arachidonic acid affects PKC in a manner consistent with activation of phospholipase A₂. These effects are cell maturation- and time-dependent and metabolite-specific. J. Cell. Physiol. 176:516–524, 1998. © 1998 Wiley-Liss, Inc.     

Effect of pertussis toxin on the phosphodiesteratic cleavage of the polyphosphoinositides by guanosine 5′-O-thiotriphosphate and thrombin in permeabilized human platelets

It has recently been shown in this laboratory that permeabilization of human platelets with 15–25 μm/ml saponin allows ADP-ribosylation by pertussis toxin of the α_i-subunit of G_i(N_i), a guanine nucleotide-binding regulatory protein. The same assay conditions have been used to determine phospholipase C in permeabilized platelets. Guanosine 5′-O-thiophosphate- (GTP[γS]-) activated phospholipase C in permeabilized platelets whose inositol phospholipids were prelabeled with [³H]inositol. Phospholipase C activity was measured by [³H]polyphosphoinositide decreases and formation of [³Hinositol bisphosphate and [³H]inositol trisphosphate. Prostacyclin, cyclic AMP or pretreatment of permeabilized platelets with pertussis toxin did not alter this effect under conditions in which the α_i-subunit was effectively ADP-ribosylated by pertussis toxin. This information indicated that ADP-ribosylation of G_i-protein was not directly related to activation or inhibition of platelet phospholipase C by GTP[γS]. Thrombin also activated phospholipase C in permeabilized platelets and, surprisingly, this action was enhanced by pertussis toxin pretreatment. This indicated that ADP-ribosylation of G_i-protein facilitates the action of thrombin on phospholipase C.     

Fatty acids and lysophospholipids as potential second messengers in auxin action. Rapid activation of phospholipase A2 activity by auxin in suspension-cultured parsley and soybean cells

Activation of cytosolic phospholipase A₂ is a typical signal transduction reaction in animal cells and occurs in plants in response to auxin, elicitors and wounding. Exogenously added fluorescent bis-BODIPY-phosphatidylcholine was taken up and hydrolysed by a cellular phospholipase A₂. Rapid activation of a phospholipase A₂ by auxin in suspension-cultured parsley ( Petrosilenum crispum L.) and soybean ( Glycine max L.) cells was shown by detection and quantification of fluorescent reaction products of phospholipase A₂. Hormone-triggered fluorescent fatty acid accumulation could be detected as early as 5 min. Auxins at 2 μM or higher concentrations activated phospholipase A₂ and fluorescent fatty acids accumulated 1.1- to threefold after 90–120 min, depending on the auxin concentration. Fluorescent lysolipid did not accumulate up to 150 μM auxin. Known inhibitors of phospholipase A₂ inhibited hormone-dependent fluorescent fatty acid accumulation in cell cultures and, previously, elongation growth in etiolated zucchini hypocotyl segments ( Scherer & Arnold (1997 ) Planta 202, 462–469). When lipids were labeled by [¹⁴C]-choline and [¹⁴C]-ethanolamine the corresponding lysophospholipids could be quantified in cell extracts. Radioactive lysophospholipids accumulated as rapidly as 1–2 min after auxin treatment but only at concentrations well above 100 μM auxin. We hypothesize that phospholipase A₂ activation is an early intermediate step between receptor and downstream responses. We hypothesize that fatty acid(s) could be second messengers in several auxin functions, especially in cell elongation. Lysophospholipids seem to be indicators or second messengers for stress caused by high auxin concentrations or may have different auxin-linked functions and are also known to accumulate during elicitor action.     

Liposomes containing alkylated methotrexate analogues for phospholipase A2 mediated tumor targeted drug delivery

Two lipophilic methotrexate analogues have been synthesized and evaluated for cytotoxicity against KATO III and HT-29 human colon cancer cells. Both analogues contained a C₁₆-alkyl chain attached to the γ-carboxylic acid and one of the analogues had an additional benzyl group attached to the α-carboxylic acid. The cytotoxicity of the γ-alkylated compound towards KATO III (IC₅₀ = 55 nM) and HT-29 (IC₅₀ = 400 nM) cell lines, was unaffected by the alkylation, whereas the additional benzyl group on the α-carboxyl group made the compound nontoxic. The γ-derivative with promising cytotoxicity was incorporated into liposomes that were designed to be particularly susceptible to a liposome degrading enzyme, secretory phospholipase A₂ (sPLA₂), which is found in high concentrations in tumors of several different cancer types. Liposome incorporation was investigated by differential scanning calorimetry (DSC), and sPLA₂ hydrolysis was examined by fluorescence spectroscopy and high performance liquid chromatography (HPLC). The results showed that the methotrexate (MTX)-analogue could be incorporated into liposomes that were degradable by sPLA₂. However, the in vitro cytotoxicity of the MTX-liposomes against KATO III and HT-29 cancer cells was found to be independent of sPLA₂ hydrolysis, indicating that the alkylated MTX-analogue was available for cancer cell uptake even in the absence of liposome hydrolysis. Using a DSC based method for assessing the anchoring stability of alkylated compounds in liposomes, it was demonstrated that the MTX-analogue partitioned into the water phase and thereby became available for cell uptake. It was concluded that liposomes containing alkylated MTX-analogues show promise as a drug delivery system, although the MTX-analogue needs to be more tightly anchored to the liposomal carrier. Also, the developed DSC-assay for studying the anchoring stability of alkylated drugs will be a useful tool in the development of liposomal drug delivery systems.