Effect Of Cis-unsaturated Fatty Acids Research Articles

Effects of cis-unsaturated fatty acids on doxorubicin sensitivity in P388/DOX resistant and P388 parental cell lines

It has been reported that several cis-unsaturated fatty acids (c-UFAs) could increase doxorubicin (DOX) accumulation in cancer cells and hence elevate its cytotoxicity. However, some researchers showed that c-UFA pretreatment did not affect its cytotoxicity in special cell lines. It is possible that the different results occurred due to different cellular characteristics. We hypothesized that c-UFA treatment might modulate the activities of some antioxidant enzymes to affect the resistance of cells to DOX. In the present study, we examined how c-UFA pretreatment affected DOX cytotoxicity on mouse leukemia cell line, P388, and its resistant subline, P388/DOX, which we found to have significantly higher glutathione peroxidase (GPx) activity as well as P-glycoprotein (p-gp) overexpression. We chose two c-UFAs, γ-linolenic acid (GLA) (18:3n-6) and docosahexaenoic acid (DHA) (22:6n-3). Cytotoxicity was measured by MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) and trypan blue exclusion assays. DOX accumulation and p-gp expression were measured by flow cytometry. The activities of catalase (CAT), superoxide dismutase (SOD), glutathione S-transferase (GST), and GPx were determined for both cell lines with and without treatment with GLA or DHA. Significant DOX accumulation occurred in both cell lines with GLA or DHA pretreatment, but without any change in p-gp expression in either cell line. Sensitivity to DOX cytotoxicity was improved by GLA or DHA pretreatment in P388/DOX in which only SOD activity was significantly increased, but not in the parental cell line P388 in which both SOD and CAT were significantly increased by the pretreatment. However, combined pretreatment of GLA or DHA with antioxidants, pyrrolidinedithiocarbamate (PDTC) or Vitamin C, could sensitize not only P388/DOX but also P388 cells to DOX. We conclude that the effects of c-UFA pretreatment on the sensitivity of cancer cells to DOX not only depend on the change in drug accumulation but also the change in the levels of antioxidant enzyme activities, and suggest that combined administration of c-UFAs, antioxidants, and DOX may be more effective in treating leukemia.

Effect of cis-unsaturated fatty acids on the activity of protein kinases and protein phosphorylation in macrophage tumor (AK-5) cells in vitro

Cis-unsaturated fatty acids (c-UFAs) have been shown to be capable of decreasing the survival of macrophage tumor (AK-5) cells in vitro. This cytotoxic action of c-UFAs was found to be associated with an increase in free radical generation and lipid peroxidation process and a simultaneous decrease in cellular anti-oxidants such as superoxide dismutase (SOD), catalase, glutathione peroxidase, glutathione reductase, glutathione and vitamin E. In the present study, it was observed that c-UFAs such as gamma linolenic acid (GLA), arachidonic acid (AA), eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) can activate phospholipase C (PLC) and enhance diacylglycerol formation; all the fatty acids except alpha linolenic acid (ALA) increased the binding of phorbol dibutyrate acetate (PDBu) suggesting translocation of protein kinase C (PKC) and at the same time these fatty acids (especially GLA, AA, EPA and DHA) also enhanced PKC activity. AA, EPA and DHA decreased the activity of protein kinase A (PKA) both in the cytosol and particulate fractions whereas ALA and GLA enhanced the PKA activity in the particulate fractions; all the fatty acids except ALA reduced cyclic AMP levels and an enhanced phosphorylation of about 13 proteins of the nuclear fraction and about eight proteins of the plasma membrane fraction was noted in c-UFA treated AK-5 cells in vitro. These results suggest that c-UFAs can alter the activities of second messenger systems such as diacylglycerol and protein kinases and can phosphorylate both plasma membrane and nuclear proteins which are likely to be components of NADPH oxidase. Based on these results, it is suggested that fatty acids may mediate their cytotoxic action in part by modulating the expression of PKC. Activated PKC may then intensify the pro-oxidant state by augmenting NADPH oxidase, so inducing superoxide anion generation which may ultimately lead to cytolysis.

Effect of cis-unsaturated fatty acids on Meth-A ascitic tumour cells in vitro and in vivo

Earlier studies performed both by us and by others have demonstrated that some n-3 and n-6 fatty acids can inhibit the growth of tumour cells in vitro. Though studies done with various types of oils rich in n-3 and n-6 fatty acids did show that the tumour incidence and growth can be modified, there were relatively few studies wherein the anti-tumour effects of individual free fatty acids were studied. Here we present results which suggest that free fatty acids (oleic acid (OA), linoleic acid (LA), α-linolenic acid (ALA), γ-linolenic acid (GLA), arachidonic acid (AA), eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA)) can inhibit the growth of methylcholanthrene-induced sarcoma cells (Meth-A cells) in vitro. The order of potency of various fatty acids on the growth of Meth-A cells was DHA>ALA>EPA>AA>GLA>LA>OA and their ID 50 values were 10, 20, 35, 45, 68, 73 and 110 μg/ml/1×10 4 cells, respectively. These results indicate that the inhibitory action of different types of n-3, n-6 and n-9 fatty acids on Meth-A cells does not depend on their unsaturation. Vitamin E could partially block the cytotoxicity of these fatty acids indicating a possible role for free radicals. GLA, AA and EPA augmented the generation of superoxide anion and lipid peroxidation in Meth-A cells indicating a possible correlation between the ability of fatty acids to augment free radicals and their tumoricidal action. In an in vivo study, it was observed that OA, LA, ALA, GLA and EPA can prolong the survival of Meth-A-bearing mice when given intraperitoneally. Of all the fatty acids tested, surprisingly, LA was found to be the most potent in enhancing the survival of the tumour-bearing animals at all the concentrations tested. Thus, these studies suggest that free fatty acids can inhibit Meth-A tumour cell proliferation both in vitro and in vivo.

Effect of cis-unsaturated fatty acids, prostaglandins, and free radicals on angiotensin-converting enzyme activity in vitro.

Angiotensin-converting enzyme (ACE) is known to play an important role in the pathobiology of human essential hypertension. Similarly, cis-unsaturated fatty acids, prostaglandins, and free radicals are believed to play a role in the control of blood pressure. It was observed that all the cis-unsaturated fatty acids tested can inhibit ACE activity to a significant degree. On the other hand, prostaglandins and free radicals, superoxide anion, hydrogen peroxide, and hydroxyl radical did not show significant inhibitory effects on ACE activity in vitro. But, the nitric oxide donor sodium nitroprusside showed a potent inhibitory action on ACE activity, suggesting that one of the possible mechanism(s) by which nitric oxide can bring about its anti-hypertensive action might be by modulating ACE activity in addition to its direct vasodilator action. These results indicate that there is a close interaction among ACE activity, cis-unsaturated fatty acids, and nitric oxide, which may have relevance to the pathobiology of human essential hypertension.

Effect of cis-unsaturated fatty acids on cellular oxidant stress in macrophage tumor (AK-5) cells in vitro.

Cis-unsaturated fatty acids (c-UFAs) induced decreased survival of macrophage tumor (AK-5) cells in vitro. The cytotoxic action of c-UfAs was associated with an increase in free radical generation and lipid peroxidation process. In addition, exposure of AK-5 cells to various c-UFAs for a short period (1 h) decreased the cellular concentrations of anti-oxidants: superoxide dismutase (SOD), catalase, glutathione peroxidase, glutathione reductase, glutathione and vitamin E. However, prolonged (24 h) exposure of AK-5 cells to c-UFAs enhanced the levels of SOD with little or no change in the concentrations of catalase, glutathione peroxidase and glutathione reductase. These results indicate that c-UFAs can enhance free radical generation and lower the concentrations of various anti-oxidants in the tumor cells which may explain the cytotoxic action of c-UFAs.

Enhancement of the Shaker B delta6-46 current by fatty acids depends on the activation of the lipoxygenase metabolic pathway.

Voltage-gated K+ channels are modulated by extracellular free unsaturated fatty acids. Both increases and attenuations of K+ channels activities have been observed. We studied the effect of cis-unsaturated fatty acids on Shaker B delta6-46 and the endogenous outward rectifier K+ channels expressed in COS cell line, using the whole cell recording technique. For both K+ channels, exogenously applied unsaturated fatty acids dramatically increased the outward K+ currents. This enhancement was not mediated by cylooxygenase or epoxygenase (P450) enzymes. However, nordihydroguaiaretic acid, a lipoxygenase metabolic pathway blocker, did prevent the arachidonic acid-induced Shaker current enhancement.

Structure-activity relationship of the effect of cis-unsaturated fatty acids on heart sarcolemmal phospholipase D activity

This study examined the role of fatty acids on the phosphatidylcholine-specific phospholipase D (PLD) function of purified sarcolemmal (SL) membranes isolated from rat hearts. The enzyme's hydrolytic activity was determined by measuring [ 14C] phosphatidic acid formation from exogenous [ 14C] phosphatidylcholine (PtdCho) in the absence or presence of the sodium salts of various saturated or unsaturated long-chain fatty acids (FA). In certain experiments the enzyme was also assayed in the transphosphatidylation mode. Cisunsaturation and free carboxyl groups were structural prerequisites for the stimulatory effect exerted by FA on SL PLD. The most effective compounds were arachidonate and oleate, which maximally activated PLD at 4 and 5 mM concentration, respectively. To verify if a detergent-like mechanism was involved in PLD activation, anionic, zwitterionic and non-ionic detergents were used. Only anionic taurodeoxycholate had a slight effect, which was about 7% of that achieved by arachidonate or oleate. These results suggest that cis-unsaturated FA activate cardiac sarcolemmal PLD by a mechanism(s) which seems to be unrelated to non-specific perturbation of the membrane.

Fatty acid homeostasis in the normoxic and ischemic heart

Fatty acid homeostasis in the normoxic and ischemic heart

Effect of cis-unsaturated fatty acids on human leukocyte cell membrane bound enzyme activity in vitro

Cis-unsaturated fatty acids (c-UFAs) are known to lower hypertension both in animals and humans. This action has been attributed to modulation of prostaglandin biosyn thesis. In essential hypertension, the activity of cell membrane bound enzyme, Na +K +-ATPase has also been shown to be altered. Here we present evidence that both n-3 and n-6 fatty acids can either augment or inhibit the activity of human leukocyte Na +K +-ATPase and 5′-Nucleotidase activities in both normals and hypertensives in vitro. Both cyclo-oxygenase and lipoxygenase inhibitors did not influence the modifying action of n-3 and n-6 fatty acids on the activity of leukocyte cell membrane bound enzymes. Fatty acid analysis of the plasma phospholipid fraction showed that the concentration of linoleic acid and arachidonic acid are low in hypertensives compared with normal controls. In addition, lipid peroxides are present in higher amounts in the plasma of hypertensives suggesting that there is an increase in free radical generation in uncontrolled essential hypertension. These results suggest that the anti-hypertensive action of cisunsaturated fatty acids can at least, in part, be due to their action on cell membrane bound enzymes, and thus free radicals and c-UFAs may have a role in essential hypertension. Thus, an understanding of the interaction between c-UFAs, free radicals, and membrane bound enzymes may be important in hypertension.

Effect of cis-unsaturated fatty acids on aortic protein kinase C activity.

Long-chain cis-unsaturated fatty acids could substitute for phosphatidylserine and activate bovine aortic protein kinase C in assays with histone as substrate. The optimal concentration was 24-40 microM for oleic, linoleic and arachidonic acids. With arachidonic acid, the Ka for Ca2+ was 130 microM and kinase activity was maximal at 0.5 mM-Ca2+. Diolein only slightly activated the oleic acid-stimulated enzyme at low physiological Ca2+ concentrations (0.1 and 10 microM). Oleic acid also stimulated kinase C activity, determined with a Triton X-100 mixed-micellar assay. Under these conditions, the fatty acid activation was absolutely dependent on the presence of diolein, but a Ca2+ concentration of 0.5 mM was still required for maximum kinase C activity. The effect of fatty acids on protein kinase C activity was also investigated with the platelet protein P47 as a substrate, since the properties of kinase C can be influenced by the choice of substrate. In contrast with the results with histone, fatty acids did not stimulate the phosphorylation of P47 by the aortic protein kinase C. Activation of protein kinase C by fatty acids may allow the selective phosphorylation of substrates, but the physiological significance of fatty acid activation is questionable because of the requirement for high concentrations of Ca2+.

Effects of long-chain cis-unsaturated fatty acids and their alcohol analogs on aggregation of bovine platelets and their relation with membrane fluidity change

The effects of long-chain cis-unsaturated fatty acids with different alkyl chain lengths and different numbers of double bonds on aggregation of bovine platelets and membrane fluidity were investigated. All the cis-unsaturated fatty acids tested inhibited aggregation and at the same time increased membrane fluidity in accordance with their inhibitory effects. The saturated fatty acids and trans-unsaturated fatty acid tested for comparison had much lower or no effects on aggregation and membrane fluidity. The inhibitory effects of mono cis-unsaturated fatty acids increased with increase of their alkyl chain length. cis-Unsaturated fatty acids with two or more double bonds had more inhibitory effects than mono-unsaturated fatty acids. The position of the double bonds had less influence than the number of double bonds. We also examined the effects of cis-unsaturated fatty acids on membrane fluidity with diphenylhexatriene and anthroyloxy derivatives of fatty acids as probes and observed increased fluidity to be considerable in the membrane. The alcohol analogs of cis-unsaturated fatty acids also inhibited aggregation and increased membrane perturbation. These results suggest that the inhibition of platelet aggregation by cis-unsaturated compounds is due to perturbation of the lipid layer.

Inhibition of platelet function by cis-unsaturated fatty acids

The uptake of free fatty acids has previously been shown to affect the capping of lymphocytes, and there is evidence that different types of fatty acids may partition into separate lipid domains in cell surface membranes. In studies of gel-filtered human platelets, we found that cis-unsaturated fatty acids (1–35 microM) inhibited platelet shape change, aggregation, and secretion of 5-hydroxytryptamine induced by thrombin, adenosine diphosphate (ADP), collagen, U46619 (a thromboxane A2 analog), or plant lectins, but not that induced by A23187, a calcium ionophore. Trans-unsaturated and saturated fatty acids had little or no inhibitory effect. The inhibitory effects of cis-unsaturated fatty acids were not affected by inhibition of adenylate cyclase or cyclooxygenase. 14C-labeled fatty acids were taken up into platelet lipids. The maximum platelet-inhibitory effect of cis-unsaturated fatty acids was seen when over 90% of the platelet label was still in the form of free fatty acids. Platelet inhibition could be reversed by washing the platelets by gel filtration. Binding of platelet agonists to the platelet was not inhibited by the fatty acids. Cis-unsaturated fatty acids, but not trans-unsaturated or saturated fatty acids, decreased fluorescence polarization of platelets or isolated platelet membranes monitored with 1,6-diphenyl- 1,3,5-hexatriene. The potency of the fatty acids as inhibitors of platelet aggregation was inversely correlated with their melting points. These data suggest that inhibition of receptor-mediated platelet responses by cis-unsaturated fatty acids results from perturbation of the platelet membrane in specific lipid domains.

Inhibition of Platelet Function by cis-Unsaturated Fatty Acids

AbstractThe uptake of free fatty acids has previously been shown to affect the capping of lymphocytes, and there is evidence that different types of fatty acids may partition into separate lipid domains in cell surface membranes. In studies of gel-filtered human platelets, we found that cis-unsaturated fatty acids (1-35 μM) inhibited platelet shape change, aggregation, and secretion of 5-hydroxytryptamine induced by thrombin, adenosine diphosphate (ADP), collagen, U46619 (a thromboxane A2 analog), or plant lectins, but not that induced by A23187, a calcium ionophore. Trans-unsaturated and saturated fatty acids had little or no inhibitory effect. The inhibitory effects of cis-unsaturated fatty acids were not affected by inhibition of adenylate cyclase or cyclooxygenase. 14C-labeled fatty acids were taken up into platelet lipids. The maximum platelet-inhibitory effect of c/s-unsaturated fatty acids was seen when over 90% of the platelet label was still in the form of free fatty acids. Platelet inhibition could be reversed by washing the platelets by gel filtration. Binding of platelet agonists to the platelet was not inhibited by the fatty acids. Cis-unsaturated fatty acids, but not trans-unsaturated or saturated fatty acids, decreased fluorescence polarization of platelets or isolated platelet membranes monitored with 1,6-diphenyl-1,3,5-hexatriene. The potency of the fatty acids as inhibitors of platelet aggregation was inversely correlated with their melting points. These data suggest that inhibition of receptor-mediated platelet responses by cis-unsaturated fatty acids results from perturbation of the platelet membrane in specific lipid domains.

Stimulation of an exocytotic event, the hamster sperm acrosome reaction, by cis-unsaturated fatty acids

This cis-unsaturated fatty acids oleic, arachidonic and cis-vaccenic stimulated the hamster sperm acrosome reaction in vitro (an exocytotic event which occurs in the sperm head and which is essential for fertilization). The trans-isomers of oleic and vaccenic acids did not stimulate the acrosome reaction, nor did the cis-unsaturated fatty acids petroselenic and docosahexaenoic or the saturated fatty acids lauric, myristic or stearic. This is the first report of a stimulatory effect of cis-unsaturated fatty acids on an exocytic event in an intact viable cell.

Modulation of membrane transport by free fatty acids: inhibition of synaptosomal sodium-dependent amino acid uptake.

High-affinity, Na+-dependent synaptosomal amino acid uptake systems are strongly stimulated by proteins which are known to bind free fatty acids. The rate of uptake as well as the overall level of accumulation is increased by such proteins as bovine serum albumin, hepatic fatty acid binding protein, beta-lactoglobulin, and fetuin. Such a stimulation is not observed with proteins which do not bind fatty acids. The transport activity of synaptosomal preparations can be directly correlated with the free fatty acid content of the preparation. Thus, incubation with albumin reduces the free fatty acid content of synaptosomal preparations, suggesting that the stimulatory effects of the proteins are related to their removal of inhibitory fatty acids formed by hydrolysis of membrane lipids during incubation. Inhibition of amino acid uptake is seen with most cis-unsaturated long chain fatty acids while saturated and trans-unsaturated fatty acids have relatively little or no effect. Under conditions in which the ionophore gramicidin D causes an increase of 22Na flux into synaptosomes, oleic acid (50 microM) has no effect on the influx. These data are consistent with the hypothesis proposed earlier by us [Rhoads, D. E., Peterson, N. A., & Raghupathy, E. (1982) Biochemistry 21, 4782] that Na+-dependent amino acid transport carrier proteins reside in a relatively fluid lipid domain in the synaptosomal membrane and that the effects of cis-unsaturated fatty acids are mediated by interactions with such domains.