Fatty acid modulation of MCF-7 human breast cancer cell proliferation,apoptosis and differentiation

Epidemiological studies suggest that dietary polyunsaturated fatty acids (PUFA) may influence breast cancer progression and prognosis. In order to study potential mechanisms of action of fatty acid modulation of tumor growth, we studied, in vitro, the influence of n-3 and n-6 fatty acids on proliferation, cell cycle, differentiation and apoptosis of MCF-7 human breast cancer cells. Both eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) inhibited the MCF-7 cell growth by 30% and 54%, respectively, while linoleic acid (LA) had no effect and arachidonic acid (AA) inhibited the cell growth by 30% (p < 0.05). The addition of vitamin E (10uM) to cancer cells slightly restored cell growth. The incubation of MCF-7 cells with PUFAs did not alter the cell cycle parameters or induce cell apoptosis. However, the growth inhibitory effects of EPA, DHA and AA were associated with cell differentiation as indicated by positive Oil-Red-O staining of the cells. Lipid droplet accumulation was increased by 65%, 30% and 15% in the presence of DHA, EPA and AA, respectively; (p < 0.05). These observations suggest that fatty acids may influence cellular processes at a molecular level, capable of modulating breast cancer cell growth.     

Vitamin C is a kinase inhibitor: dehydroascorbic acid inhibits IkappaBalpha kinase beta

Reactive oxygen species (ROS) are key intermediates in cellular signal transduction pathways whose function may be counterbalanced by antioxidants. Acting as an antioxidant, ascorbic acid (AA) donates two electrons and becomes oxidized to dehydroascorbic acid (DHA). We discovered that DHA directly inhibits IkappaBalpha kinase beta (IKKbeta) and IKKalpha enzymatic activity in vitro, whereas AA did not have this effect. When cells were loaded with AA and induced to generate DHA by oxidative stress in cells expressing a constitutive active IKKbeta, NF-kappaB activation was inhibited. Our results identify a dual molecular action of vitamin C in signal transduction and provide a direct linkage between the redox state of vitamin C and NF-kappaB signaling events. AA quenches ROS intermediates involved in the activation of NF-kappaB and is oxidized to DHA, which directly inhibits IKKbeta and IKKalpha enzymatic activity. These findings define a function for vitamin C in signal transduction other than as an antioxidant and mechanistically illuminate how vitamin C down-modulates NF-kappaB signaling.     

Cellular pathways for transport and efflux of ascorbate and dehydroascorbate

The mechanisms allowing the cellular transport of ascorbic acid represent a primary aspect for the understanding of the roles played by this vitamin in pathophysiology. Considerable research effort has been spent in the field, on several animal models and different cell types. Several mechanisms have been described to date, mediating the movements of different redox forms of ascorbic acid across cell membranes. Vitamin C can enter cells both in its reduced and oxidized form, ascorbic acid (AA) and dehydroascorbate (DHA), utilizing respectively sodium-dependent transporters (SVCT) or glucose transporters (GLUT). Modulation of SVCT expression and function has been described by cytokines, steroids and post-translational protein modification. Cellular uptake of DHA is followed by its intracellular reduction to AA by several enzymatic and non-enzymatic systems. Efflux of vitamin C has been also described in a number of cell types and different pathophysiological functions were proposed for this phenomenon, in dependence of the cell model studied. Cellular efflux of AA is mediated through volume-sensitive (VSOAC) and Ca²⁺-dependent anion channels, gap-junction hemichannels, exocytosis of secretory vesicles and possibly through homo- and hetero-exchange systems at the plasma membrane level. Altogether, available data suggest that cellular efflux of ascorbic acid - besides its uptake - should be taken into account when evaluating the cellular homeostasis and functions of this important vitamin.     

Inhibition of copper-induced LDL oxidation by vitamin C is associated with decreased copper-binding to LDL and 2-oxo-histidine formation

Oxidatively modified low-density lipoprotein (LDL) has numerous atherogenic properties, and antioxidants that can prevent LDL oxidation may act as antiatherogens. We have previously shown that vitamin C (L-ascorbic acid, AA) and its two-electron oxidation product dehydro-L-ascorbic acid (DHA) strongly inhibit copper (Cu)-induced LDL oxidation. These findings are unusual, as AA is known to act not only as an antioxidant, but also a pro-oxidant in the presence of transition metal ions in vitro, and DHA has no known reducing capacity. Here we report that human LDL (0.4 mg protein/ml) incubated with 40 μM Cu²⁺ binds 28.0 ± 3.3 Cu ions per LDL particle (mean ± SD, n = 10). Co-incubation of LDL with AA or DHA led to the time- and concentration-dependent release of up to 70% of bound Cu, which was associated with the inhibition of LDL oxidation. Incubation of LDL with Cu and AA or DHA also led to the time-dependent formation of 2-oxo-histidine, an oxidized derivative of histidine with a low affinity for Cu. Addition of free histidine prevented the formation of the LDL-Cu complexes and inhibited LDL oxidation, despite the fact that Cu remained redox-active. Interestingly, histidine was more effective than AA or DHA at limiting Cu binding to LDL, but at low concentrations AA and DHA were more effective than histidine at inhibiting LDL oxidation. These data suggest that there are at least two types of Cu binding sites on LDL: those that bind Cu in a redox-active form critical for initiation of LDL oxidation, and those that bind Cu in a redox-inactive form not contributing to LDL oxidation. The former sites may be primarily histidine residues of apolipoprotein B-100 that are oxidized to 2-oxo-histidine in the presence of Cu and AA or DHA, thus explaining, at least in part, the unusual inhibitory effect of vitamin C on Cu-induced LDL oxidation.     

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 vitamin C and vitamin E on prostaglandin synthesis by fibroblasts and squamous carcinoma cells.

Dietary levels of vitamins C and E have been associated with cancer prevention and to a lesser extent with therapeutic enhancement of cancer treatment. Inhibition of prostaglandins (PGs) by pharmacological agents has been demonstrated to enhance immunocompetence, and to suppress growth of tumors in animals and humans. We report here on the effect of vitamins C and E on PGE2 production by human gingival fibroblasts and SCC-25 oral squamous carcinoma cells. The results indicate: 1. vitamins C and E exert a dose-dependent effect on arachidonic acid (AA) release and PGE2 synthesis; 2. vitamin E has a biphasic effect which is stimulatory at 1 and 10 microM and inhibitory at 100 microM; 3. vitamin E is considerably more potent than vitamin C in its inhibitory effect on AA and PGE2 in both cell types; 4. a combination of the two vitamins has a consistent dose-dependent inhibitory effect on AA and PGE2; 5. vitamin C stimulates PGE2 synthesis from exogenous AA in fibroblasts, and inhibits it in SCC-25 cells. The in vivo significance of these findings requires further investigation.     

Docosahexaenoic acid induces proteasome-dependent degradation of estrogen receptor α and inhibits the downstream signaling target in MCF-7 breast cancer cells

About two thirds of breast cancers in women are hormone-dependent and require estrogen for growth, its effects being mainly mediated through estrogen receptor α (ERα). Docosahexaenoic acid (DHA, 22:6n-3) and arachidonic acid (AA, 20:4n-6) have opposite effects on carcinogenesis, with DHA suppressing and AA promoting tumor growth both in vitro and in vivo. However, the mechanism is not clear. Here, we examined whether the effect is mediated through changes in ERα distribution. MCF-7 cells, an ERα-positive human breast cancer cell line, was cultured in estrogen-free medium containing 0, 10 or 60 μM DHA or AA, then were stimulated with estradiol. DHA supplementation resulted in down-regulation of ERα expression (particularly in the extranuclear fraction), a reduction in phosphorylated MAPK, a decrease in cyclin D1 levels and an inhibition in cell viability. In contrast, AA had no such effects. The DHA-induced decrease in ERα expression resulted from proteasome-dependent degradation and not from decreased ERα mRNA expression. We propose that breast cancer cell proliferation is inhibited by DHA through proteasome-dependent degradation of ERα, reduced cyclin D1 expression and inhibition of MAPK signaling.     

2-O-α-D-Glucopyranosyl-l-ascorbic acid as an antitumor agent for infusion therapy

Ascorbic acid (AA) has been reported as a treatment for cancer patients. Intravenous (iv) administration of high-dose AA increases plasma AA levels to pharmacologic concentrations and generates reactive oxygen species (ROS) to exert anti-tumor activity via enhancement of oxidative stress. However, AA is very unstable in aqueous solutions and it is impossible to preserve AA for a long period in a solution. 2-O-α-D-Glucopyranosyl-l-ascorbic acid (AA-2G), which is a glucoside derivative of AA, has been found to exhibit much higher stability than AA in aqueous solutions and it shows vitamin C activity after enzymatic hydrolysis to AA. To evaluate the effectiveness of AA-2G for cancer treatment, we examined the antitumor activity of AA-2G to murine colon carcinoma (colon-26) cells and in tumor-bearing mice. AA-2G did not show cytotoxicity to colon-26 cells, whereas AA exhibited a significant cytotoxic effect in a concentration-dependent manner. In colon-26 tumor-bearing mice, iv administration of high-dose AA-2G as well as that of AA significantly inhibited tumor growth. Experiments on the biodistribution and clearance of AA-2G in tumor-bearing mice showed that AA-2G was rapidly hydrolyzed to AA and exhibited significant antitumor activity. Treatment of tumor-bearing mice with AA-2G tended to increase plasma malondialdehyde level. These results indicated that the antitumor activity of AA-2G was caused by ROS generated by AA released by rapid hydrolysis of AA-2G.     

Docosahexaenoic acid inhibits invasion of human RT112 urinary bladder and PT45 pancreatic carcinoma cells via down-modulation of granzyme B expression

Fish oil-derived n-3 polyunsaturated fatty acids (n-3 PUFAs) inhibit invasion of some tumor cell types in vitro and in vivo. The mechanisms underlying this activity are unclear. Here, we examined the capability of n-3 PUFA-docosahexaenoic acid (22:6n-3; DHA) to affect the invasiveness of human RT112 urinary bladder and PT45 pancreatic carcinoma cell lines in vitro and the mechanism underlying this activity; n-6 PUFA-arachidonic acid (20:4n-6; AA) served as control. We showed that, in contrast to AA, 25, 50 and 100 μM DHA significantly inhibited in a dose-dependent manner the invasion through Matrigel of both RT112 and PT45 cells. Then, we analyzed whether the serine proteinase granzyme B (GrB), originally known as cytotoxic molecule of lymphocytes and recently also characterized for its extracellular functions such as invasion promotion of bladder cancer cells, might be involved in the invasion inhibitory activity exerted by DHA. We demonstrated that, accordingly to RT112 bladder cancer cells, PT45 cells expressed GrB and GrB promoted their invasion, since stealth RNA interference-mediated down-regulation of GrB dramatically suppressed PT45 cell invasion. Notably, we also showed that, in contrast to AA, 25, 50 and 100 μM DHA induced a dose-dependent down-modulation of GrB expression in both RT112 and PT45 cells. In conclusion, DHA can reduce the invasive phenotype of bladder and pancreatic carcinoma cells, and we provide the first evidence for a possible causative role of GrB in DHA-induced inhibition of cancer cell invasion. The potential use of fish oil as adjuvant antibladder and antipancreatic cancer agent may be suggested.     

Arachidonic and docosahexaenoic acids differentially affect the expression of fatty acyl-CoA oxidase, protein kinase C and lipid peroxidation in HepG2 cells.

Arachidonic (AA) and docosahexaenoic (DHA) acids (5-20 microM), when supplemented to human hepatoma HepG2 cells, which are depleted in these long-chain polyunsaturated fatty acids in conventional culture conditions, enhance the expression of acyl-CoA oxidase (ACOX), the first enzyme in the peroxisomal beta-oxidation cycle. DHA is effective at lower concentrations (at 5 microM) and to a greater extent (about 60% increment) than AA (about 40%) at 20 microM. Protein kinase C (PKC) appears to be involved in the activity of AA on ACOX, but not in that of DHA, since only the effect of AA is prevented by the PKC inhibitor Staurosporine, and since a remarkable elevation of the PKC activator diacylglycerol occurs only after AA supplementation. AA also induces elevation of lipoperoxides, favoured by the relative vitamin E deficiency occurring in cultured cells, and this effect, which is prevented by supplementation of the vitamin, may contribute to PKC activation.     

Ascorbic acid depletion enhances expression of the sodium-dependent vitamin C transporters, SVCT1 and SVCT2, and uptake of ascorbic acid in livers of SMP30/GNL knockout mice

In this study, we examined whether ascorbic acid (AA) and dehydroascorbic acid (DHA), the oxidized form of AA, levels in tissues regulate the AA transporters, sodium-dependent vitamin C transporters (SVCT) 1 and SVCT2 and DHA transporters, glucose transporter (GLUT) 1, GLUT3, GLUT4 mRNA by using senescence marker protein-30 (SMP30)/gluconolactonase (GNL) knockout (KO) mice. These mice are incapable of synthesizing AA in vivo. AA depletion enhanced SVCT1 and SVCT2 mRNA expression in the liver and SVCT1 and GLUT4 mRNA expression in the small intestine, but not in the cerebrum or kidney. Next, we examined the actual impact of AA uptake by using primary cultured hepatocytes from SMP30/GNL KO mice. In the AA-depleted hepatocytes from SMP30/GNL KO mice, AA uptake was significantly greater than in matched cultures from wild-type mice. These results strongly affirm that intracellular AA is an important regulator of SVCT1 and SVCT2 expression in the liver.     

Plasma membrane gamma-glutamyltransferase activity facilitates the uptake of vitamin C in melanoma cells

Adequate cellular transport of ascorbic acid (AA) and its oxidation product dehydroascorbate (DHA) is assured through specific carriers. It was shown that vitamin C is taken up as DHA by most cell types, including cancer cells, via the facilitative GLUT transporters. Thus, AA oxidation to DHA can be considered a mechanism favoring vitamin C uptake and intracellular accumulation. We have investigated whether such an AA-oxidizing action might be provided by plasma membrane gamma-glutamyltransferase (GGT), previously shown to function as an autocrine source of prooxidants. The process was studied using two distinct human metastatic melanoma clones. It was observed that the Me665/2/60 clone, expressing high levels of membrane GGT activity, was capable of effecting the oxidation of extracellular AA, accompanied by a marked increase of intracellular AA levels. The phenomenon was not observed with Me665/2/21 cells, possessing only traces of membrane GGT. On the other hand, AA oxidation and stimulation of cellular uptake were indeed observed after transfection of 2/21 cells with cDNA coding for GGT. The mechanism of GGT-mediated AA oxidation was investigated in acellular systems, including GGT and its substrate glutathione. The process was observed in the presence of redox-active chelated iron(II) and of transferrin or ferritin, i.e., two physiological iron sources. Thus, membrane GGT activity-often expressed at high levels in human malignancies-can oxidize extracellular AA and promote its uptake efficiently.     

Ascorbic acid suppresses cell death in rat DS-sarcoma cancer cells induced by 5-aminolevulinic acid-based photodynamic therapy

Especially in the public, vitamin C is considered supportive for the treatment of cancer and supplementation is common. However, the underlying mechanism that most chemotherapeutic agents, ionizing radiation, and photodynamic therapy exert on tumor cell kill is an increased production of reactive oxygen species (ROS) leading to irreversible tissue injury. Therefore, antioxidants like ascorbic acid (AA) may prevent cancer cells of cellular free radical damage and may therefore be contraindicated in patients undergoing tumor treatment. We report on the effects of AA on markers of oxidative stress and apoptosis in rat DS-sarcoma cells on 5-aminolevulinic acid-based photodynamic therapy (ALA-PDT). AA dose-dependently protected cancer cells against lipid and protein oxidation caused by ALA-PDT treatment. By real-time RT-PCR analysis an impressive increase of FasL (124-fold) and TNF-alpha (121-fold) mRNA was detected after PDT treatment. In addition, a decrease in mitochondrial transmembrane potential followed by the mitochondrial release of apoptosis-inducing factor (AIF) was observed. All these early signs of apoptosis were significantly reduced by AA, resulting in a 2.1-fold increased cell survival rate on ALA-PDT treatment. In conclusion, AA functions as a potent antioxidant, protecting mitochondria and other cell structures of oxidative cell injury induced by ALA-PDT and may therefore be contraindicated in patients undergoing tumor treatment.     

Oxidative stress-mediated antitumor activity of erythorbic acid in high doses

Intravenous (iv) infusion of high-dose ascorbic acid (AA) has been used as a treatment for cancer patients. The tumoricidal action of AA occurs due to its prooxidant effect. Erythorbic acid (EA), one of the AA epimers, has reduced vitamin C activity, while the antioxidant activity of EA is similar to that of AA. Currently, other physiological and pharmacological functions of EA are not well known. We examined the cytotoxicity of EA to murine colon carcinoma (colon-26) cells and the antitumor activity of EA in tumor-bearing mice. Cytotoxic activity of EA to colon-26 cells was evaluated by using the calcein-AM assay. EA showed the same cytotoxic activity to colon-26 cells as that of AA. The cytotoxicity of EA was shown to be caused by oxidative stress. Next, colon-26 tumor-bearing mice were iv administered EA and AA on alternate days for 4 times, and tumor growth rates were measured. Tumor growth was significantly inhibited by administration of high-dose EA in vivo as well as AA. Finally, the in vivo biodistribution and clearance of EA and AA were investigated in tumor-bearing mice. Endogenous AA in the tumor was consumed to resist oxidative stress caused by reactive oxygen species that was generated by administered EA. These results indicated that the oxidative stress-mediated antitumor activity is one of the pharmacological functions of high-dose iv EA.     

Suppression of platelet-activating factor generation and modulation of arachidonate metabolism by dietary enrichment with (n-9) eicosatrienoic acid or docosahexaenoic acid in mouse peritoneal cells

Several studies have shown that dietary n-3 polyunsaturated fatty acids (PUFAs) suppress platelet-activating factor (PAF) generation in leukocytes of humans and rodents, which is associated with the antagonism of arachidonic acid metabolism. Dietary eicosatrienoic acid (20:3n-9, ETrA) is also suggested to antagonize arachidonic acid (AA) metabolism, but its effect on PAF generation in leukocytes has not been defined. In the present study, we investigated the effects of an ETrA-rich diet on PAF generation and AA metabolism in mouse peritoneal cells, which were compared with those of a docosahexaenoic acid (DHA)-rich diet. Mice were fed a diet supplemented with a lipid preparation rich in ETrA, a DHA-rich fish oil (FO) or palm oil (PO) for 3 weeks, and peritoneal cells containing more than 80% of monocytes/macrophages were obtained. The peritoneal cells in the DHA and ETrA diet groups generated upon zymosan stimulation a smaller amount of PAF than cells in the PO diet group. In the peritoneal cells of the DHA diet group, AA contents in phosphatidylcholine (PC) and phosphatidylethanolamine (PE) were significantly lower than those in cells of the PO diet group, but those in phosphatidylinositol (PI) were not significantly different between the two dietary groups. A considerable amount of ETrA was incorporated into the peritoneal cells of the ETrA diet group, and AA was reduced as compared with the PO diet group. These changes occurred preferentially in PI but to a less extent in PC and PE. The amount of free AA released by the peritoneal cells upon zymosan stimulation was significantly reduced in the DHA diet group as compared with that in the PO diet group, whereas AA release was similar between the PO and ETrA diet groups. In conclusion, the effects of dietary ETrA on AA content in the phospholipid subclasses and AA release were quite different from those of dietary DHA, although both diets suppressed PAF generation in mouse peritoneal cells to a similar extent.