Synthesis and anti-tumour, immunomodulating activity of diosgenin and tigogenin conjugates.

A series of novel diosgenin (DSG) and tigogenin (TGG) derivatives with diosgenin or tigogenin steroid aglycons linked to levulinic and 3,4-dihydroxycinnamic acids, dipeptides and various amino acids by an ester bond at the C3-oxygen atom of the steroid skeleton has been synthesized. Diosgenyl esters have been prepared by an esterification reaction (DCC/DMAP) of diosgenin with the corresponding acids. All analogues have been evaluated in vitro for their antiproliferative profile against cancer cell lines (MCF-7, MDA-MB-231, PC-3) and human umbilical vein endothelial cells (HUVEC). Analogue2c (l-serine derivative of TGG), the best representative of the series showed IC50 of 1.5 µM (MCF-7), and induced apoptosis in MCF-7 by activating caspase-3/7. The immunomodulatory properties of six synthesized analogues have been determined by examining their effects on the expression of cytokine genes essential for the functioning of the human immune system (IL-1, IL-4, IL-10, IL-12 and TNF-α). Biological evaluation has revealed that new compounds 4c and 16a do not induce the expression of pro-inflammatory cytokines in THP-1 cells after the lipopolysaccharide (LPS) stimulation. They also stimulate the expression of anti-inflammatory IL-10 that acts stronger than diosgenin itself. An in silico ADME properties(absorption, distribution, metabolism, excretion) study was also performed to predict the pharmacokinetic profile of the synthesized compounds. To shed light on the molecular interactions between the synthesized compounds and the glucocorticoid receptor and the estrogen receptor, 2c, 4c and 16a compounds were docked into the active binding sites of these receptors. The in silico and in vitro data suggested that this new group of compounds might be considered as a promising scaffold for further modification of more potent and selective anticancer and immunomodulatory agents.

Phosphatidylserine biosynthesis in mitochondria from Ehrlich ascites tumor cells.

1. Isolated mitochondria of Ehrlich ascites tumor cells incorporated [14C]serine into phosphatidylserine and partly into its decarboxylation product, phosphatidylethanolamine, while phosphatidylserine was the sole product with microsomes. 2. The incorporation of [14C]serine into mitochondrial phospholipids was stimulated by Ca2+ which indicated the operation of the Ca2+-dependent base-exchange mechanism, virtually absent in mammalian tissue mitochondria. The finding cannot be attributed to microsomal contamination. 3. The incorporation of [14C]serine into mitochondrial phospholipids was also stimulated by ATP, both in the presence and in the absence of calcium-complexing agents. The stimulation by ATP was insensitive to penicillin and streptomycin, thus pointing that this process was not of bacterial origin. 4. The latter process was further stimulated by phosphatidic acid and phosphatidic acid precursors, but not by CDP diacylglycerol. 32P from neither [gamma-32P]ATP nor [32P]phosphatidic acid was incorporated into phosphatidylserine in the presence of CTP and L-serine. The release of [14C]CMP from [14C]CDP diacylglycerol was not stimulated by l-serine. 5. It is concluded that [14C]serine incorporation into mitochondrial phospholipids by ATP-dependent process does not fit to any of the pathways of phopholipid biosynthesis described so far.

Transfer of phosphatidic acid between microsomal and mitochondrial outer and inner membranes.

A protein fraction from rat liver cytoplasm, precipitable at 50-95% saturation of ammonium sulphate, binds phosphatidic acid from mitochondrial and microsomal membranes. Protein-bound phosphatidic acid was eluted from Sephadex G-75 in fractions corresponding to a molecular weight of about 10 000. No such binding was observed with mitochondrial soluble proteins, either total or precipitated with ammonium sulphate between 50 and 95% saturation. The transfer of phosphatidic acid from microsomes to mitochondria was increased by liver cytoplasmic proteins precipitable at 50-95% saturation of ammonium sulphate but not with mitochondrial soluble proteins. This increase by cytoplasmic proteins was pronounced in 200 mM sucrose but was negligible in 100 mM KCI where the spontaneous transfer was quite high. Cytoplasmic proteins stimulated the synthesis of cardiolipin and phosphatidylglycerol in mitochondria deprived of the outer membrane but not in intact mitochondria when phosphatidic acid was supplied either by microsomes or liposomes. It is suggested that the transfer of phosphatidic acid from the outer to the inner mitochondrial membrane is not mediated by transfer proteins but occurs either by direct contact of the membranes or as free diffusion through the aqueous phase.

Transport of phosphatidic acid within the mitochondrion.

Transfer of phosphatidic acid from the outer to the inner membrane within intact rat liver mitochondria was assessed by measuring the ratio of lipid 32P to the marker enzyme of the outer membrane, rotenone-insensitive NADH-cytochrome c reductase, in the outer and inner membrane fractions obtained after incubation of mitochondria under conditions for net synthesis of [32P]phosphatidic acid. This transfer was found to proceed with time, to occur only under high ionic strength of the external medium and to be insensitive to N-ethylmaleimide and factors reducing the number of contact sites between the two mitochondrial membranes. These results are interpreted as supporting the idea that phosphatidic acid transport within the mitochondrion occurs as free diffusion through the aqueous phase and not being mediated by phospholipid transfer protein(s).

Role of lamellar bodies in the biosynthesis of phosphatidylcholine in mouse lung.

1. A lamellar body-enriched fraction was isolated from whole lung homogenates of mouse lung and its contamination with microsomes, mitochondria, and cytosol protein assessed by marker enzyme analyses. 2. By measuring the activity of cholinephosphotransferase (EC 2.7.8.2) in varying amounts of microsomes in the presence and absence of a fixed quantity of lamellar bodies, it could be demonstrated unequivocally that lamellar bodies of mouse lung lack the capacity to synthesize phosphatidylcholine de novo. 3. A similar approach allowed the conclusion that lamellar bodies of mouse lung do not contain lysophosphatidylcholine acyltransferase (EC 2.3.1.23) and lysophosphatidylcholine:lysophosphatidylcholine acyltransferase (EC 2.3.1.--), enzymes which play a putative role in the formation of pulmonary 1,2-dipalmitoyl-sn-glycerol-3-phosphocholine. The activities of these enzymes observed in lamellar body fractions could be attributed completely to contaminating microsomes and cytosol respectively. 4. Lamellar bodies contributed to the activity of microsomal lysophosphatidylcholine acyltransferase by a cooperative effect. The possible role of this cooperation in the biosynthesis of 1,2-dipalmitoyl-sn-glycero-3-phosphocholine is discussed.

Synthesis of phospholipids in mitochondria and other membrane fractions of rabbit reticulocytes.

1. Reticulocytosis of 40-50% was obtained in rabbits by daily bleeding. Reticulocytes (plus erythrocytes) were subfractionated into plasma membrane fraction, mitochondria and the post-mitochondrial fraction. 2. In all fractions, fatty acids were incorporated into phospholipids. This process was ATP dependent and represented acylation of lysophospholipids. 3. Incorporation of fatty acids into lysophosphatidic and phosphatidic acids occurred only in the presence of sn-glycerol 3-phosphate and was observed in mitochondria and the post-mitochondrial fraction. It represents a two-step acylation of sn-glycerol 3-phosphate. 4. Incorporation of phosphorylcholine from CDPcholine into phosphatidylcholine was observed in the mitochondrial and the post-mitochondrial fractions. This activity was correlated with NADPH-cytochrome c reductase and was probably connected with the remnants of the endoplasmic reticulum.

Different regulation of phospholipase D activity in glioma C6 cells by sphingosine, propranolol, imipramine and phorbol ester.

In has been found that sphingosine, propranolol, imipramine and phorbol ester (12-O-tetradecanoylphorbol-13-acetate, TPA) have a stimulatory effect on phospholipase D activity in glioma C6 cells. The cells were prelabelled with [1-(14)C]palmitic acid and phospholipase D-mediated synthesis of [(14)C]phosphatidylethanol was measured. The enhancing effect of TPA was almost completely blocked by a specific protein kinase C inhibitor, GF 109203X. In contrast, GF 109203X failed to inhibit the sphingosine, imipramine and propranolol stimulatory effects, indicating that their stimulation was independent of protein kinase C. The effect of TPA on phospholipase D was also blocked by imipramine and propranolol, whereas sphingosine additively potentiated TPA-mediated phospholipase D activity, both at shorter and longer (2-60 min) times of incubation. These results suggest that in glioma C6 cells, sphingosine is not only involved in a different phospholipase D activation than the TPA regulatory system, but also that it operates in a different compartment of the cell.

Changes in Ca2+ concentration in phorbol ester and thapsigargin treated glioma C6 cells. The role of protein kinase C in regulation of Ca2+ entry.

Glioma C6 cells treated with 12-0-tetradecanoyl-phorbol-13-acetate, TPA (10 nM and 100 nM) manifested slow increase in intracellular calcium concentration ([Ca2+]i), dependent upon both Ca2+ release from intracellular stores and Ca2+ entry, and ranging from 50 to 500 nM in different cells. The effect of TPA was abolished by the down-regulation procedure and by protein kinase C inhibitors, such as staurosporine (100 nM), suramin (100 microM), and sphingosine (100 microM), pointing to a role of protein kinase C (PKC) in this process. On the other hand, thapsigargin (100 nM), a selective inhibitor of the endoplasmic reticulum Ca(2+)-ATPase, produced a rapid increase in [Ca2+]i (up to 800 nM). This increase consisted of a transient initial phase followed by sustained elevation in [Ca2+]i, typical of Ca2+ release from intracellular stores and of Ca2+ entry, respectively. However, when the cells were exposed to TPA (100 nM) prior to thapsigargin (100 nM), then thapsigargin produced only a transient rise in [Ca2+]i. We suggest that TPA, a PKC activator, affects thapsigargin-induced Ca2+ entry, probably by PKC-mediated changes in cytoskeleton structures.

Mechanism of the ATP-dependent phosphatidylserine synthesis in liver subcellular fractions.

It has been shown that the ATP-dependent incorporation of [14C]serine into phosphatidylserine in rat liver mitochondrial and microsomal fractions is prevented by EGTA. On the other hand, at low (microM) Ca2+ concentrations, serine incorporation is strongly stimulated by ATP and Mg2+. This stimulatory effect is reduced by calcium ionophore A23187. It is therefore suggested that the ATP-dependent process is that of serine base-exchange reaction, stimulated by endogenous Ca2+ accumulated inside the microsomal vesicles by Ca2+,Mg2+-ATPase. The mitochondrial activity can be accounted for by contamination by the endoplasmic reticulum.

A new pathway for phosphatidylserine synthesis in rat liver microsomes.

It has been shown that the phosphate moiety of glycerophosphate is incorporated into phosphatidylserine of rat liver microsomes, but not of mitochondria. The reaction is dependent on CMP. This observation suggests that the new pathway of acyl-specific synthesis of phosphatidylserine proposed by J.P. Infante [(1984) FEBS Lett. 170, 1-14] can proceed in rat liver microsomes.

Serine base-exchange in rat liver nuclei.

It has been shown that the incorporation of [(14)C]serine into phosphatidylserine (PS) in isolated rat liver nuclei is intrinsic to this organelle as attested by marker enzyme activity. Serine incorporation into PS was the highest in nuclei depleted of the outer membrane of the nuclear envelope (nucleoplasts) and negligible in the outer membrane. Trypsin treatment of nucleoplasts caused a strong inactivation of PS synthesis and only a moderate one of the NAD pyrophosphorylase activity, the marker enzyme of the inner nuclear membrane. We suggest that the serine base-exchange enzyme is located in the inner membrane of the nuclear envelope and accessible from the periplasmic surface of this membrane.

Two subtypes of G protein-coupled nucleotide receptors, P2Y(1) and P2Y(2) are involved in calcium signalling in glioma C6 cells.

1. In glioma C6 cells, the stimulation of P2Y receptors by ADP, ATP and UTP initiated an increase in the intracellular Ca2+ concentration, in a process that involved the release of Ca2+ from InsP(3)-sensitive store and the capacitative, extracellular Ca2+ entry. The presence of external Ca2+ was not necessary to elevate Ca(2+). 2. The rank order of potencies of nucleotide analogues in stimulating [Ca2+](i) was: 2MeSADP > ADP > 2MeSATP = 2ClATP > ATP > UTP. alpha,beta-Methylene ATP, adenosine and AMP were ineffective. 3. ADP and UTP effects were additive, while actions of ATP and UTP were not additive on [Ca2+](i) increase. Similarly, cross-desensitization between ATP and UTP but not between ADP and UTP occurred. 4. Suramin, a non-specific nucleotide receptors inhibitor, antagonized ATP-, UTP- and ADP-evoked Ca2+ responses. PPADS, a selective antagonist of the P2Y(1) receptor-generated InsP(3) accumulation, decreased ADP-initiated Ca2+ response with no effect on ATP and UTP. 5. Pertussis toxin (PTX) reduced ADP- and ATP-induced Ca2+ increases. Short-term treatment with TPA, inhibited both ATP and ADP stimulatory effects on [Ca2+](i). 6. ADP inhibited isoproterenol-induced cyclic AMP accumulation. PTX blocked this effect, but PPADS did not. 7. RT - PCR analysis revealed the molecular identity of P2Y receptors expressed by glioma C6 cells to be both P2Y(1) and P2Y(2). 8. It is concluded that both P2Y(1) and P2Y(2) receptors co-exist in glioma C6 cells. ADP acts as agonist of the first, and ATP and UTP of the second one. Both receptors are linked to phospholipase C (PLC).

Intracellular Ca2+ signals induced by ATP and thapsigargin in glioma C6 cells. Calcium pools sensitive to inositol 1,4,5-trisphosphate and thapsigargin.

In glioma C6 cells, extracellular ATP generates inositol 1,4,5-trisphosphate (InsP3), indicating the presence of purinergic receptors coupled to phosphoinositide turnover. To identify the effect of ATP (acting via InsP3) and thapsigargin (acting without InsP3 production as a specific inhibitor of the endoplasmic reticulum Ca(2+)-ATPase) on intracellular Ca2+ pools we used video imaging of Fura-2 loaded into single, intact glioma C6 cells. It has been shown that ATP and thapsigargin initiate Ca2+ response consistent with the capacitative model of Ca2+ influx. When the cells were stimulated by increasing concentrations of ATP (1, 10, 50 and 100 microM) the graded, quantal Ca2+ response was observed. In the absence of extracellular Ca2+ thapsigargin and ionomycin-releasable Ca2+ pools are overlapping, demonstrating that Ca2+ stores are located mainly in the endoplasmic reticulum. After maximal Ca2+ mobilization by ATP, thapsigargin causes further increase in cytosolic Ca2+ concentration, whereas emptying of thapsigargin-sensitive intracellular stores prevents any further Ca2+ release by ATP. Thus, the thapsigargin-sensitive intracellular pool of Ca2+ in glioma C6 cells seems to be larger than that sensitive to InsP3. Two hypothesis to explain this result are proposed. One postulates a presence of two different Ca2+ pools, sensitive and insensitive to InsP3 and both discharged by thapsigargin, and the other, the same intracellular pool of Ca2+ completely emptying by thapsigargin and only partially by InsP3. These results may contribute to understanding the mechanism of Ca2+ signalling mediated by ATP, the most potent intracellular Ca2+ mobilizing agonist in all types of glial cells.

Effect of ethanol on ATP-induced phospholipases C and D and serine base exchange in glioma C6 cells.

The effect of extracellular ATP, a nucleotide receptor agonist in the central nervous system, was investigated in glioma C6 cells on the intracellular Ca2+ level and the formation of phosphatidylethanol and phosphatidic acid in the presence and absence of ethanol (150 mM). In the cells prelabeled with [14C]palmitic acid, 100 microM ATP induced both the hydrolysis and the transphosphatidylation reactions leading to the formation of [14C]phosphatidic acid; addition of ethanol generated [14C]phosphatidylethanol. However, ATP-mediated increase in the level of [14C]phosphatidic acid was not inhibited by ethanol. Furthermore, ethanol augmented ATP-induced transient and sustained increase in the intracellular Ca2+ concentration, whereas ethanol alone did not produce any change in the intracellular Ca2+ level. These results indicate that in glioma C6 cells, ATP induces activation of polyphosphoinositide-specific phospholipase C and phospholipase D and that ethanol enhances this effect. In the present investigation we have also shown that long-term (2 days) ethanol treatment, at concentration relevant to chronic alcoholism (100 mM), decreased the incorporation of [14C]serine into phosphatidylserine. Since the effect of ethanol on ATP-induced activities of phospholipase C and phospholipase D and on serine base-exchange in glioma C6 cells differs significantly from that in cultured neuronal cells, these results may contribute to a better understanding of the mechanisms of ethanol action in cells of glial origin.

Lipids and signal transduction in the nucleus.

During the last few years a growing amount of data has accumulated showing phospholipid participation in nuclear signal transduction. Very recent data strongly support the hypothesis that signal transduction in the nucleus is autonomic. Local production of inositol polyphosphates, beginning with the activation of phospholipase C is required for their specific function in the nucleus. Enzymes which modify polyphosphoinositols may control gene expression. Much less information is available about the role of other lipids in nuclear signal transduction. The aim of this minireview is to stress what is currently known about nuclear lipids with respect to nuclear signal transduction.

Sphingosine, sphingosylphosphorylcholine and sphingosine 1-phosphate modulate phosphatidylserine homeostasis in glioma C6 cells.

The effect of sphingosine, sphingosylphosphorylcholine and sphingosine 1-phosphate on L-[U-14C]serine incorporation into phosphatidylserine and phosphatidylserine-derived phosphatidylethanolamine was investigated in intact glioma C6 cells. Sphingosine, sphingosylphosphorylcholine and sphingosine 1-phosphate are potent signalling molecules which, due to their physicochemical features, may function as amphiphilic compounds. It has been found that sphingosine and sphingosylphosphorylcholine (amphiphilic cations) significantly increase [14C]phosphatidylserine synthesis and decrease the amount of 14C-labeled phosphatidylethanolamine. Sphingosine 1-phosphate (an amphiphilic anion) was without effect on phosphatidylserine synthesis but, similarly as sphingosine and sphingosylphosphorylcholine, reduced the conversion of phosphatidylserine to phosphatidylethanolamine. These results strongly suggest that sphingosine, sphingosylphosphorylcholine and sphingosine 1-phosphate can modulate cellular phospholipid homeostasis by stimulation of phosphatidylserine synthesis and an interference with phosphatidylserine decarboxylase.

Exogenous sphingosine 1-phosphate and sphingosylphosphorylcholine do not stimulate phospholipase D in C6 glioma cells.

In the present study we investigate the effect of exogenous sphingosine, sphingosine 1-phosphate and sphingosylphosphorylcholine on phospholipase D (PLD) activity in glioma C6 cells. The cells were prelabeled with [1-14C]palmitic acid and PLD-mediated synthesis of [14C]phosphatidylethanol was measured. Sphingosine 1-phosphate and sphingosylphosphorylcholine did not stimulate [14C]phosphatidylethanol formation either at low (0.1-10 microM) or high (25-100 microM) concentrations. On the other hand, sphingosine at concentrations of 100-250 microM strongly stimulated PLD activity as compared to the effect of phorbol ester, 12-O-tetradecanoylphorbol 13-acetate (TPA), known as a PLD activator. The effect of TPA on PLD is linked to the activation of protein kinase C. The present study also shows that sphingosine additively enhances TPA-mediated PLD activity. This is in contrast to the postulated role of sphingosine as a protein kinase C inhibitor. These results demonstrate that in glioma C6 cells sphingosine not only affects PLD independently of its effect on protein kinase C, but also is unable to block TPA-mediated PLD activity.

Oligosaccharide organization of the beta-subunits of pig kidney Na+/K+-ATPase.

The distance between the beta-subunits of Na+/K+-ATPase isolated from pig dark red kidney medulla was determined by Förster energy transfer. First, oligosaccharides of the beta-subunit were shown to be labelled with three fluorophores: Lucifer yellow (LY), Lissamine rhodamine B sulfonyl hydrazine (LRSH) and Cascade blue (CB). Further, LY and LRSH were used as the donor and the acceptor, respectively, for Förster energy transfer studies to determine the localization of the beta-subunit in the native enzyme which is known to be formed as a tetramer (alphabeta)2. It was found that the beta-subunits in the functional enzyme complex in the membrane are not localized next to each other but are spatially separated. The distance between fluorophores covalently attached to the beta-subunits was found to be 5.1 nm. This conclusion was confirmed by measurements with another donor-acceptor pair CB-LY. The results also support the idea of a direct interaction of the beta-subunit with the extracellular part of the alpha-subunit. These interactions were modified in the presence of millimolar concentrations of magnesium ions. This indicates a crucial role of magnesium in extracellular interactions between the alpha and beta subunits.

Sphingosine stimulates calcium mobilization and modulates calcium signals evoked by thapsigargin in glioma C6 cells.

The effect of sphingosine on intracellular calcium signalling in glioma C6 cells was studied with Fura-2 video imaging technique. Sphingosine had a direct effect on changes in cytosolic Ca2+ concentration only when applied at high concentration of 100 microM, causing the cytosolic Ca2+ level to rise. However, at a much lower concentration of 15 microM sphingosine diminished calcium responses triggered by thapsigargin (a specific inhibitor of calcium pump in the endoplasmic reticulum) and ionomycin (calcium ionophore). Since responses to thapsigargin and ionomycin were blocked in Ca(2+)-free medium, we postulate that sphingosine is acting on the intracellular calcium stores. Additionally, sphingosine (at 15 microM and 100 microM) markedly decreases thapsigargin-induced sustained elevation in cytosolic Ca2+ concentration, indicating its inhibitory effect on thapsigargin-evoked Ca2+ influx. Sphingosine is a known inhibitor of protein kinase C and the involvement of this enzyme is postulated in the modulatory effects of sphingosine on intracellular calcium dynamics.

Temporal dynamics and regional distribution of [14C]serine uptake into mouse brain.

In order to examine the uptake of L-serine into brain structures and brain metabolic compartments, L-[U-14C]serine was injected into tail vein of mice. The uptake was examined 30 min, 90 min, 3 h and 5 h after injection by both quantitative autoradiography of coronal brain sections and by biochemical analysis. Brain radioactivity was extracted and partitioned into protein associated pellets, metabolites soluble in aqueous phase and lipids soluble in the organic phase. Most of the radioactivity was found in the aqueous phase, about 10% was incorporated into lipids. Among phospholipids the highest label was found in phosphatidylserine, then in phosphatidylethanolamine and in phosphatidylcholine, it amounted to 52%, 30% and 18% of label by 90 min after injection, respectively. The brain distribution of L-serine uptake resembled that described for strychnine-insensitive [3H]glycine binding, with cortical structures being preferentially labelled.