Antitumor effect of oleic acid; mechanisms of action: a review.

INTRODUCTION: The beneficial effects of oleic acid in cancer processes can no longer be doubted, but little is known about the mechanisms of action behind this phenomenon. AIM: The aim of the present review is to clarify whether oleic acid has an effect on important mechanisms related to the carcinogenic processes. METHODS: We searched electronic databases and bibliographies of selected articles were inspected for further reference. We focused our research on two cellular transformations characterizing cancer development: proliferation and cell death or apoptosis. RESULTS: Numerous studies have reported an inhibition in cell proliferation induced by oleic acid in different tumor cell lines. Herein, oleic acid could suppress the over-expression of HER2 (erbB-2), a well-characterized oncogene which plays a key role in the etiology, invasive progression and metastasis in several human cancers. In addition, oleic acid could play a role in intracellular calcium signaling pathways linked to the proliferation event. Regarding cell death, oleic acid has been shown to induce apoptosis in carcinoma cells. The mechanisms behind the apoptotic event induced by oleic acid could be related to an increase in intracellular ROS production or caspase 3 activity. Several unsaturated fatty acids have been reported to induce apoptosis through a release of calcium from intracellular stores. However, evidence regarding such a role in oleic acid is lacking. CONCLUSIONS: Oleic acid plays a role in the activation of different intracellular pathways involved in carcinoma cell development. Such a role could be the root of its antitumoral effects reported in clinical studies.

Arachidonic acid inhibits capacitative calcium entry in rat thymocytes and human neutrophils.

Emptying the intracellular Ca2+ stores by treatment with the endomembrane Ca2+-ATPase inhibitor thapsigargin activates capacitative Ca2+ entry (CCE). This can be evidenced in fura-2-loaded cells by an increase of [Ca2+]i or by an acceleration of Mn2+ entry. Micromolar concentrations of arachidonic acid inhibited CCE induced by treatment with thapsigargin in rat thymocytes and in human neutrophils. This inhibitory action was shared by other unsaturated fatty acids, but not by the saturated arachidic acid nor by arachidonic acid methyl ester. The effect was not due to metabolites derived from arachidonic acid since several non-metabolizable analogs were able to reproduce it. Phorbol dibutyrate (PDB) acted similarly, suggesting that the inhibitory effect could be mediated by activation of protein kinase C (PKC). However, whereas the inhibition of CCE by PDB was reversed by treatment with the PKC inhibitor staurosporin, the inhibition by arachidonic acid was not. We find that unsaturated fatty acids antagonized microsomal dealkylation of benzyl-resorufin, a cytochrome P450-mediated activity, with the same specificity profile as for inhibition of CCE. These results are consistent with previous proposals suggesting that a microsomal cytochrome P450 may be involved in the regulation of CCE.

Functional ATP receptors in rat anterior pituitary cells.

The effects of ATP and other nucleotides on the cytosolic Ca2+ concentration ([Ca2+]i) of single immunocytochemically typed anterior pituitary (AP) cells have been studied. ATP increased [Ca2+]i in a large percentage (60-88%) of all five AP cell types: lactotropes, somatotropes, corticotropes, gonadotropes, and thyrotropes. Additivity experiments suggest the presence of at least two different receptors, one accepting both ATP and UTP (U receptor), producing Ca2+ release from the intracellular stores, and the other preferring ATP (A receptor), producing Ca2+ (and Mn2+) entry. The characteristics of the U and A receptors were consistent with those of P2Y2 and P2X2, respectively, and their distribution in the different AP cell types was not homogeneous. The presence of other ATP receptors such P2Y1 or P2X2/P2X3 heteropolymers in a small fraction of the cells cannot be excluded. Thus functional ionophoric P2X receptors, which are typical of neural tissue, are also present in the pituitary gland and could contribute to regulation of the gland's function.

Nitric oxide synthesis in rat peritoneal macrophages is induced by IgE/DNP complexes and cyclic AMP analogues. Evidence in favor of a common signaling mechanism.

The production of nitric oxide was studied in rat adherent peritoneal cells stimulated with preformed IgE/DNP-BSA complexes made of IgE obtained from a secreting hybridoma. Stimulation with complexes at equivalence induced both the production of NO and an increased expression of the mRNA of the inducible isoform of NO synthase (iNOS). Production of NO was also elicited by a rabbit polyclonal F(ab')2 anti-CD23 cross-reacting with rat CD23. Because IgE/DNP-BSA complexes did not elicit Ca2+ mobilization and genistein did not influence the production of NO, cyclic AMP was considered as an alternative signaling molecule. Combination of a suboptimal concentration of dibutyryl cyclic AMP and IgE/DNP-BSA complexes showed an additive effect on NO production, whereas this was not observed when the agonists were used at supraoptimal doses. The inhibitor of cyclic AMP-specific phosphodiesterase IV, rolipram, which acts on the enzyme isoform predominantly expressed in inflammatory cells, also induced the production of NO. Furthermore, IgE/DNP-BSA complexes increased intracellular levels of cyclic AMP. Taken together, these data indicate that stimulation of mononuclear phagocytes via the low-affinity receptor Fc epsilon RII or rising intracellular concentrations of cyclic AMP leads to an enhanced expression of iNOS. Evidence in favor of the involvement of cyclic AMP in the signaling pathway linked to Fc epsilon RII is provided by the effect of IgE/DNP-BSA complexes on intracellular cyclic AMP levels and by the additive effect produced by dibutyryl cyclic AMP on NO production elicited by IgE/DNP-BSA complexes.

Rapid refilling of Ca2+ stores in macrophages stimulated by ATP involves the sequential activation of phospholipase D and protein kinase C.

Ca2+ movements between intracellular stores, the cytoplasm and external solution were analysed in murine peritoneal macrophages stimulated by various agonists. The Ca2+ content of intracellular stores was estimated from the amplitude of Ca(2+)-transients elicited by ionomycin applied in Ca(2+)-free solution. Both uridine 5'-triphosphate (UTP) and platelet-activating factor (PAF) triggered the release of Ca2+ followed by a sustained influx, during which intracellular stores remained totally empty. In contrast, in the continuous presence of adenosine 5'-triphosphate (ATP), Ca2+ was initially released and then rapidly sequestered again by the stores. ATP-induced store refilling was not related to cell depolarization or to an increase in the intracellular Na+ concentration (two specific consequences of ATP stimulation which are not induced by PAF and UTP). Store refilling was not caused by a signal that ATP would fail to induce (e.g. as a result of receptor desensitization), but was positively controlled by ATP, even in the simultaneous presence of a concentration of PAF which, on its own, would have caused a persistent store depletion. The hypothesis that the signal delivered by ATP involves the sequential activation of phospholipase D and protein kinase C is consistent with the present pharmacological evidence. However, although we found conditions in which Ca2+ stores did not refill in the presence of ATP, this maintained store depletion was not accompanied by a sustained Ca2+ response similar to that elicited by PAF or UTP, suggesting that store depletion is a condition which is necessary, but not sufficient, for inducing Ca2+ influx.

Rotavirus interaction with isolated membrane vesicles.

To gain information about the mechanism of epithelial cell infection by rotavirus, we studied the interaction of bovine rotavirus, RF strain, with isolated membrane vesicles from apical membrane of pig enterocytes. Vesicles were charged with high (quenching) concentrations of either carboxyfluorescein or calcein, and the rate of fluorophore release (dequenching) was monitored as a function of time after mixing with purified virus particles. Purified single-shelled particles and untrypsinized double-shelled ones had no effect. Trypsinized double-shelled virions induced carboxyfluorescein release according to sigmoid curves whose lag period and amplitude were a function of virus concentration and depended on both temperature and pH. The presence of 100 mM salts (Tris Cl, NaCl, or KCl) was required, since there was no reaction in isoosmotic salt-free sorbitol media. Other membrane vesicle preparations such as apical membranes of piglet enterocyte and rat placenta syncytiotrophoblasts, basolateral membranes of pig enterocytes, and the undifferentiated plasma membrane of cultured MA104 cells all gave qualitatively similar responses. Inhibition by a specific monoclonal antibody suggests that the active species causing carboxyfluorescein release is VP5*. Ca2+ (1 mM), but not Mg2+, inhibited the reaction. In situ solubilization of the outer capsid of trypsinized double-shelled particles changed release kinetics from sigmoidal to hyperbolic and was not inhibited by Ca2+. Our results indicate that membrane destabilization caused by trypsinized outer capsid proteins of rotavirus leads to fluorophore release. From the data presented here, a hypothetical model of the interaction of the various states of the viral particles with the membrane lipid phase is proposed. Membrane permeabilization induced by rotavirus may be related to the mechanism of entry of the virus into the host cell.

The pathway for refilling intracellular Ca2+ stores passes through the cytosol in human leukaemia cells.

The pathway for refilling the intracellular Ca2+ stores of HL60 and U937 human leukaemia cells loaded with fura-2 has been investigated. On addition of external Ca2+ to cells with empty stores there was an increase in the cytosolic Ca2+ concentration ([Ca2+]i) which preceded the refilling of the stores. The increase in [Ca2+]i was faster than the refilling, by 3- to 15-fold, depending on the cell type. In measurements in single HL60 cells we found that the refilling of the stores correlated with the extent of the [Ca2+]i increase on addition of external Ca2+. The cells showing no [Ca2+]i increase were unable to refill their stores. The addition of Ni2+ to the extracellular medium prevented both the [Ca2+]i increase and the refilling of the stores. These results indicate that the limiting step for store refilling is the entry of Ca2+ from the extracellular medium to the cytosol. Hence, we conclude that extracellular Ca2+ cannot gain access directly to the intracellular Ca2+ stores in these cells, but must first enter the cytosol and be taken up from there into the stores.

Calcium responses elicited by nucleotides in macrophages. Interaction between two receptor subtypes.

The responses elicited by ATP and UTP in macrophages (measured by microfluorescence and in patch-clamp) present marked differences. The release of Ca2+ from intracellular stores induced by ATP is due to the activation of P2U receptors. These receptors can be activated by ATP4- and by MgATP2-, with apparent K0.5 values of 0.65 and 6.5 microM, respectively. The release of Ca2+ due to activation of P2U receptors by either ATP or UTP is followed by the opening of ionic channels leading to an influx of Ca2+. A second pathway for Ca2+ influx results from the opening of P2Z receptor channels triggered by adenosine-5'-O(1-thiotriphosphate) or ATP but not by UTP. The form of ATP that activates P2Z receptors is ATP4- (with a K0.5 of 0.5 microM). In voltage-clamped cells, the inward current activated by ATP4- is transient, partly because it inactivates and partly because it is rapidly masked by the development of a quinine-sensitive Ca(2+)-dependent K+ current. In current-clamp, macrophages stimulated by UTP remain normally polarized, whereas ATP depolarizes them. This P2Z-mediated depolarization results in an inhibition of the influx of Ca2+, which explains part of the difference between the time courses of the Ca2+ responses elicited by ATP and UTP.

Control of Ca2+ entry into HL60 and U937 human leukaemia cells by the filling state of the intracellular Ca2+ stores.

Differentiation of HL60 cells by treatment with dimethyl sulphoxide induces the expression of membrane receptors for N-formylmethionyl-leucyl-phenylalanine (fMLP) and for platelet-activating factor (PAF). In these cells both agonists produced an increase in the cytosolic Ca2+ concentration ([Ca2+]i) by release of Ca2+ from the intracellular stores, followed shortly by an acceleration of the entry of Ca2+ or Mn2+, used here as a Ca2+ surrogate for Ca2+ channels. Cytochrome P-450 inhibitors blocked the agonist-induced entry of Ca2+ or Mn2+ with no modification of Ca2+ release from the stores. Emptying the intracellular Ca2+ stores either by treatments inducing no inositol phosphate production, such as prolonged incubation in Ca(2+)-free medium or treatment with the Ca2+ ionophore ionomycin, increased the plasma-membrane permeability to Ca2+ and Mn2+. This Ca(2+)-store-regulated Mn2+ entry was inhibited by Ni2+ and by cytochrome P-450 inhibitors. Refilling of the Ca2+ stores by incubation in Ca(2+)-containing medium restored low Mn2+ permeability. The same mechanism is present and functional in non-differentiated cells, before expression of membrane receptors for fMLP and PAF. These results suggest that agonist-induced Ca2+ (Mn2+) entry is secondary to the emptying of the intracellular Ca2+ stores, which in turn activates plasma-membrane channels by a mechanism involving cytochrome P-450.

Heterogeneity of L-alanine transport systems in brush-border membrane vesicles from rat placenta during late gestation.

The placental uptake of L-alanine was studied by using purified brush-border membrane vesicles from rat trophoblasts. Saturation curves were carried out at 37 degrees C in buffers containing 100 mM (zero-trans)-NaSCN, -NaCl, -KSCN, -KCl, or -N-methyl-D-glucamine gluconate. The uncorrected uptake results were fitted by non-linear regression analysis to an equation involving one diffusional component either one or two saturable Michaelian transport terms. In the presence of NaCl, two distinct L-alanine transport systems were distinguished, named respectively System 1 (S-1; Vm1 about 760 pmol/s per mg of protein; KT1 = 0.5 mM) and System 2 (S-2; Vm2 about 1700 pmol/s per mg; KT2 = 9 mM). In contrast, in the presence of K+ (KCl = KSCN) or in the absence of any alkali-metal ions (N-methyl-D-glucamine gluconate), only one saturable system was apparent, which we identify as S-2. When Na+ is present, S-1, but not S-2, appears to be rheogenic, since its maximal transport capacity significantly increases in the presence of an inside-negative membrane potential, created either by replacing Cl- with the permeant anion thiocyanate (NaSCN > NaCl) or by applying an appropriate K+ gradient and valinomycin. alpha-(Methylamino)isobutyrate (methyl-AIB) appears to be a substrate of S-1, but not of S-2. For reasons that remain to be explained, however, methyl-AIB inhibits S-2. We conclude that S-1 represents a truly Na(+)-dependent mechanism, where Na+ behaves as an obligatory activator, whereas S-2 cannot discriminate between Na+ and K+, although its activity is higher in the presence of alkali-metal ions than in their absence (Na+ = K+ > N-methyl-D-glucammonium ion). S-2 appears to be fully developed 2 days before birth, whereas S-1 undergoes a capacity-type activation between days 19.5 and 21.5 of gestation, i.e. its apparent Vmax. nearly doubles, whereas its KT remains constant.