Differential anti-cancer effects of purified EPA and DHA and possible mechanisms involved.

As the concepts of pharmaconutrition are receiving increasing attention, it seems essential to clearly assess the effects of specific dietary compounds in specific groups of patients or clinical conditions. We are herein interested in better defining the differential anti-neoplastic effects of the two major n-3 long chain polyunsaturated fatty acids present in fish oil, eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA). The efficiency of these fatty acids represents a subject of intense interest and debate, and whereas plenty of preclinical studies have strongly demonstrated their preventive and therapeutic effect in different kinds of cancers, the results of the epidemiologic studies are still controversial, and only a few trials have been performed. It has been reported that EPA and DHA may act either through the same or different mechanisms, thus suggesting that a differential efficacy could exist. At present, however, this point has not been clarified, although its better comprehension would allow a more proper and effective use of these fatty acids in the human interventional studies. In an attempt to elucidate this aspect we have herein analyzed the data obtained in the studies which have directly compared the antitumor effects of separate treatments with EPA or DHA. Most of the in vitro data indicate DHA as the more powerful antineoplastic agent. However, an equivalent efficiency of EPA and DHA is suggested by the few in vivo studies. Possible reasons for this discrepancy are discussed and pathways of cell growth that could be differentially influenced by EPA and DHA are described.

Chemoprevention of lung pathologies by dietary n-3 polyunsaturated fatty acids.

Different intervention trials have been so far conducted and others are ongoing to evaluate the effect of increased intake of n-3 polyunsaturated fatty acids (PUFAs) in the prevention of several disorders affecting lungs and airways. They have been focused on chronic obstructive pulmonary disease, acute respiratory distress syndrome, acute lung injury, pulmonary fibrosis, alteration of lung function in cystic fibrosis, as well as asthma and cachexia in lung cancer patients. Their outcomes are not always consistent, but, if beneficial effects were observed, they have been related mainly to the anti-inflammatory action of n-3 PUFAs. On the contrary, trials investigating their effects on the development and progression of lung cancer are still not available. This in spite of the huge number of equivalent studies performed on other kind of cancers (breast, colon and prostate cancer), which share with lung cancer the highest incidence in Western countries and an elevated sensitivity to chemoprevention. Contrasting results were also obtained by the few epidemiological studies available on lung cancer. However, different experimental studies, performed in vivo and in vitro, provided strong indications of the anti-tumor action of n-3 PUFAs against lung cancer, and identified molecular mechanisms for their action. In this review our effort will concentrate in critically reviewing the current evidence for the beneficial effect of n-3 PUFAs in inflammatory and neoplastic disorders of lungs and airways, and in identifying possible molecular mechanisms underlying their effects.

Dietary n-3 PUFA vascular targeting and the prevention of tumor growth and age-related macular degeneration.

The protective role of dietary n-3 polyunsaturated fatty acids (PUFAs) against cardiovascular diseases has been partly related to their ability to modulate the risk condition known as "endothelial dysfunction", by reverting the endothelial alterations associated to it (reduced vascular reactivity, the proinflammatory state, and the prothrombotic properties). Moreover, vasculature represents the target for inhibition of pathologic neo-angiogenesis by n-3 PUFAs. This effect is believed to contribute to the beneficial action of these fatty acids against disorders which recognize neovascularization as a crucial pathogenetic step for their development, such as cancer and age-related macular degeneration (AMD). Many epidemiological studies have been conducted to evaluate the association between the intake of these fatty acids and the risk of developing cancer or AMD, even though contrasting and not definitive results have been obtained. Conversely, plenty of preclinical and in vitro experimental studies have provided evidence for the anti-angiogenic effects of n-3 PUFAs, mainly studying neo-angiogenesis in general (using normal endothelial cells in vitro) or as a step of cancer growth. The main aim of this review is to critically review the current evidence for the inhibition of the neo-angiogenic process exerted by n-3 PUFAs in cancer and AMD, and to identify possible molecular mechanisms that might contribute to their beneficial effects.

Antineoplastic effects of n-3 polyunsaturated fatty acids in combination with drugs and radiotherapy: preventive and therapeutic strategies.

Many data support the beneficial effect of n-3 polyunsaturated fatty acids (PUFAs) as chemopreventive and chemotherapeutic agents in the treatment of several chronic pathologies including cancer. Different molecular mechanisms have been proposed to explain their effects, including alterations in arachidonic acid oxidative metabolism and metabolic conversion of n-3 PUFAs to novel discovered bioactive derivatives; modification of oxidative stress; changes in cell membrane fluidity and structure and altered metabolism and function of membrane proteins. Considerable knowledge has been recently gathered on the possible beneficial effects of n-3 PUFAs administered in combination with different antineoplastic drugs and radiotherapy against melanoma, leukemia, neuroblastoma, and colon, breast, prostate, and lung cancer. The efficacy of these combinations has been demonstrated both in vivo and in vitro, and clinical trials have also been conducted. The aim of this review is to analyze all the n-3 PUFA combinations investigated so far, their efficacy, and the possible molecular mechanisms involved. It would be highly auspicable that the detailed analysis of the literature in this field could further support the common use of n-3 PUFAs in combination with other chemopreventive agents and warrant more clinical investigations designed to test the effectiveness of n-3 PUFA treatments coupled with conventional antineoplastic therapies.

N-3 polyunsaturated fatty acid effect in periodontal disease: state of art and possible mechanisms involved.

Anti-inflammatory properties have been widely reported for n-3 polyunsaturated fatty acids (PUFAs) and some studies have been focalized on their possible role in the modulation of gingivitis and alveolar bone resorption in periodontal disease (PD). Increased formation of arachidonic acid-derived inflammatory eicosanoids and augmented oxidative stress are two molecular mechanisms pathogenetically involved in the progression of PD and known to be inhibited by n-3 PUFAs in PD setting. The present review will focus also on other molecular pathways and factors known to be altered in the development of PD and known to be subject to n-3 PUFA modulation in other pathological settings different from PD. Overall, the available findings strongly encourage further experimental studies on animals subject to experimental PD and treated with n-3 PUFAs, long term n-3 PUFA intervention studies on PD patients and molecular studies to identify additional potential molecular routes of n-3 PUFA action in PD.

n-3 polyunsaturated fatty acids and the prevention of colorectal cancer: molecular mechanisms involved.

Increasing evidence supports the hypothesis that nutrition habits play a critical role in the incidence and growth of colorectal cancer. Among dietary factors, fish-derived n-3 polyunsaturated fatty acids (PUFAs) have gained particular interest, since epidemiological studies have shown a reduced incidence of this cancer in populations consuming high levels of fish. Also a variety of experimental studies and different clinical trials substantiated the beneficial role of n-3 PUFAs. Such an anti-neoplastic activity has been related to the regulatory effects exhibited by n-3 PUFAs on cell proliferation and apoptosis. Anti-angiogenic and anti-metastatic effects have been also reported for these fatty acids. Finally, it has been suggested that they may act as adjuvant therapeutic agents sensitizing tumors, including colon cancer, to different anti-neoplastic drugs. Several molecular mechanisms have been hypothesized to explain their anti-neoplastic action and, in particular, the modulating effect on the expression of several proteins involved in the regulation of cell cycle and apoptosis, such as Bcl-2, Bax, c-Myc seem to play a central role. Their inhibitory action has been also recently suggested for the molecular pathways driven by COX-2 and beta-catenin, known to play a major role in the development and progression of colon cancer. The aim of the present review is to analyze the anti-neoplastic effect of n-3 PUFAs towards colon cancer, and examine the molecular mechanisms involved.

Dual role of beta-carotene in combination with cigarette smoke aqueous extract on the formation of mutagenic lipid peroxidation products in lung membranes: dependence on pO2.

Results from some intervention trials indicated that supplemental beta-carotene enhanced lung cancer incidence and mortality in chronic smokers. The aim of this study was to verify the hypothesis that high concentrations of the carotenoid, under the pO2 present in lung (100-150 mmHg), may exert deleterious effects through a prooxidant mechanism. To test this hypothesis, we examined the interactions of beta-carotene and cigarette smoke condensate (tar) on the formation of lipid peroxidation products in rat lung microsomal membranes enriched in vitro with varying beta-carotene concentrations (from 1 to 10 nmol/mg prot) and then incubated with tar (6-25 microg/ml) under different pO2. As markers of lipid peroxidation, we evaluated the levels of conjugated dienes and malondialdehyde, possessing mutagenic and pro-carcinogenic activity. The exposure of microsomal membranes to tar induced a dose-dependent enhancement of lipid peroxidation, which progressively increased as a function of pO2. Under a low pO2 (15 mmHg), beta-carotene acted clearly as an antioxidant, inhibiting tar-induced lipid peroxidation. However, the carotenoid progressively lost its antioxidant efficiency by increasing pO2 (50-100 mmHg) and acted as a prooxidant at pO2 ranging from 100 to 760 mmHg in a dose-dependent manner. Consistent with this finding, the addition of alpha-tocopherol (25 microM) prevented the prooxidant effects of the carotenoid. beta-Carotene auto-oxidation, measured as formation of 5,6-epoxy-beta,beta-carotene, was faster at high than at low pO2 and the carotenoid was more rapidly consumed in the presence of tar. These data point out that the carotenoid may enhance cigarette smoke-induced oxidative stress and exert potential deleterious effects at the pO2 normally present in lung tissue.

Lycopene induces apoptosis in immortalized fibroblasts exposed to tobacco smoke condensate through arresting cell cycle and down-regulating cyclin D1, pAKT and pBad.

There is a lot of interest in the health benefits of dietary carotenoids and on the relationship of these compounds with smoke. In particular, it is unknown if the enhanced cancer risk observed in smokers following beta-carotene supplementation can be also found using other carotenoids. Here, we studied the effects of the tomato carotenoid lycopene on molecular pathways involved in cell cycle progression, apoptosis and survival in immortalized RAT-1 fibroblasts exposed to cigarette smoke condensate (TAR). Lycopene (0.5-2.0 microM) inhibited cell growth in a dose-and time-dependent manner, by arresting cell cycle progression and by promoting apoptosis in cells exposed to TAR. The arrest of cell cycle was independent of p53 and of 8-OH-dG DNA damage and related to a decreased expression of cyclin D1. Moreover, the carotenoid up-regulated apoptosis and down-regulated the phosphorylation of AKT and Bad in cells exposed to TAR. Such an effect was associated to an inhibition of TAR-induced expression of Cox-2 and hsp90, which is known to maintain AKT activity. This study suggests that lycopene, differently from beta-carotene, can exert protective effects against cigarette smoke condensate.

Dietary alpha-linolenic acid reduces COX-2 expression and induces apoptosis of hepatoma cells.

Fatty acid synthetase (FAS) is overexpressed in various tumor tissues, and its inhibition and/or malonyl-CoA accumulation have been correlated to apoptosis of tumor cells. It is widely recognized that both omega-3 and omega-6 polyunsaturated fatty acids (PUFAs) depress FAS expression in liver, although epidemiological and experimental reports attribute antitumor properties only to omega-3 PUFA. Therefore, we investigated whether lipogenic gene expression in tumor cells is differently regulated by omega-6 and omega-3 PUFAs. Morris hepatoma 3924A cells were implanted subcutaneously in the hind legs of ACI/T rats preconditioned with high-lipid diets enriched with linoleic acid or alpha-linolenic acid. Both-high lipid diets depressed the expression of FAS and acetyl-CoA carboxylase in tumor tissue, this effect correlating with a decrease in the mRNA level of their common sterol regulatory element binding protein-1 transcription factor. Hepatoma cells grown in rats on either diet did not accumulate malonyl-CoA. Apoptosis of hepatoma cells was induced by the alpha-linolenic acid-enriched diet but not by the linoleic acid-enriched diet. Therefore, in this experimental model, apoptosis is apparently independent of the inhibition of fatty acid synthesis and of malonyl-CoA cytotoxicity. Conversely, it was observed that apoptosis induced by the alpha-linolenic acid-enriched diet correlated with a decrease in arachidonate content in hepatoma cells and decreased cyclooxygenase-2 expression.

Redox regulation of cell proliferation by pyrrolidine dithiocarbamate in murine thymoma cells transplanted in vivo.

Pyrrolidine dithiocarbamate (PDTC) is a synthetic compound largely used in cell biological studies and known to exert either antioxidant or pro-oxidant effects. Recently, its antitumoral activity has been proposed on the basis of its antioxidant and proapoptotic effects. In the present study, we evaluated the effect of increasing i.p. doses of PDTC on the growth of a strain of highly malignant thymoma cells inoculated in the peritoneum of inbred Balb/c mice. PDTC treatment increased the number of thymoma cells in a dose-dependent manner, enhancing the percentage of proliferating tumor cells. PDTC exerted regulatory effects on cell cycle distribution, decreasing the expression of cell cycle inhibitors. Alterations in the production of intracellular reactive oxygen species, levels of oxidized glutathione, and intracellular levels of the redox-active metals iron and copper were also observed. The above results represent the first evidence that PDTC may induce in vivo cell proliferation in a murine thymoma cell model. In addition, we suggest that the ability of PDTC to bind and transport metals inside the cell and its pro-oxidant property may be factors underlying its effects on thymoma cell proliferation and cell cycle distribution.

beta-carotene at high concentrations induces apoptosis by enhancing oxy-radical production in human adenocarcinoma cells.

This is the first report demonstrating a relationship between apoptosis induction and changes of intracellular redox potential in the growth-inhibitory effects of high concentrations of beta-carotene in a tumor cell line. beta-Carotene inhibited the growth of human WiDr colon adenocarcinoma cells in a dose- and time-dependent manner, induced apoptosis, and blocked Bcl-2 expression. These effects were accompanied by an enhanced production of intracellular reactive oxygen species (ROS). The addition of the antioxidant alpha-tocopherol blocked both the pro-oxidant and the growth-inhibitory effects of the carotenoid. These findings suggest that beta-carotene may act as an inductor of apoptosis by its pro-oxidant properties.

Mitogenic and apoptotic signaling by carotenoids: involvement of a redox mechanism.

The potential for carotenoids to modulate tumor growth is currently under investigation. Although epidemiological studies evidence that a high intake of vegetables, rich in carotenoids, decreases cancer incidence and mortality, clinical trials demonstrate that supplementation of beta-carotene to chronic smokers or to asbestos workers increases the risk for lung cancer. These contradictory findings have renewed interest in elucidating the mechanism of action of carotenoids in biological systems. In this review, we show evidence for mitogenic and apoptotic effects of carotenoids and we support the hypothesis that these molecules may act as anticarcinogens or as procarcinogens through a redox mechanism. In particular, we report demonstrations for the anti-oxidant or pro-oxidant effects of carotenoids in vitro and in vivo, focusing our attention on the relationship existing between cell growth and redox status.

Novel antioxidant agents deriving from molecular combinations of vitamins C and E analogues: 3,4-dihydroxy-5(R).

Molecular combinations of two antioxidants (i.e., ascorbic acid and the pharmacophore of alpha-tocopherol), namely the 2,3-dihydroxy-2,3-enono-1,4-lactone and the chromane residues, have been designed and tested for their radical scavenging activities. When evaluated for their capability to inhibit malondialdehyde (MDA) production in rat liver microsomal membranes, the 3,4-dihydroxy-5R-2(R,S)-(6-hydroxy-2,5,7,8-tetramethylchroman-2(R,S)yl-methyl)-1,3]dioxolan-4S-yl]-5H-furan-2-one (11a-d), exhibited an interesting activity. In particular the 5R,2R,2R,4S and 5R,2R,2S,4S isomers (11c,d) displayed a potent antioxidant effect compared to the respective synthetic alpha-tocopherol analogue (5) and natural alpha-tocopherol or ascorbic acid, used alone or in combination. Moreover, the mixture of stereoisomers 11a-d also proved to be effective in preventing damage induced by reperfusion on isolated rabbit heart, in particular at the higher concentration of 300 microM. In view of these results our study represents a new approach to potential therapeutic agents for applications in pathological events in which a free radical damage is involved. Design, synthesis and preliminary biological activity are discussed.

Canthaxanthin supplementation alters antioxidant enzymes and iron concentration in liver of Balb/c mice.

The 4,4'-diketo-beta-carotene, canthaxanthin, alters tocopherol status when fed to Balb/c mice, suggesting an involvement of carotenoids in the modulation of oxidative stress in vivo. We investigated further the modifications induced by an oral administration of canthaxanthin on lipid peroxidation, antioxidant enzymes and iron status in liver of Balb/c mice. Female 6-wk-old Balb/c mice were randomly divided into two groups (n = 10/group). The control group (C) received olive oil alone (vehicle) and the canthaxanthin-treated group (Cx) received canthaxanthin at a dose of 14 microg/(g body wt.d). The 15-d canthaxanthin treatment resulted in carotenoid incorporation but did not modify lipid peroxidation as measured by endogenous production of malondialdehyde (MDA). However, glutathione peroxidase activity was 35% lower (P<0.01) and catalase (59%, P<0.005) and manganese superoxide dismutase (MnSOD) (28%, P<0.05) activities were higher in canthaxanthin-treated mice than in controls. Moreover, carotenoid feeding caused a significant (P<0.05) overexpression of the MnSOD gene; mRNA levels of the enzyme were greater in treated mice than in controls. Concomitantly, a 27% (P<0.05) greater iron concentration was found in liver from canthaxanthin-treated mice compared with controls. These findings support the hypothesis that canthaxanthin alters the protective ability of tissues against oxidative stress in vivo.

n-3 PUFA dietary supplementation inhibits proliferation and store-operated calcium influx in thymoma cells growing in Balb/c mice.

The antitumor effect of daily individual administration of eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) (2 g/kg body weight) in Balb/c mice bearing a transplantable thymoma was investigated. Mice received oleic acid (control group), EPA and DHA ethyl esters starting 10 days before tumor inoculation. Analysis of phospholipid composition of neoplastic cell revealed that EPA and DHA levels were significantly increased (63 and 22% increase) after EPA and DHA treatments, respectively. Conversely, decreased levels of arachidonic acid were found in both cases (19 and 24% decrease in EPA and DHA groups, respectively). EPA and DHA delayed the appearance of macroscopic ascites (100% of animal, from 7 to 28 days), prolonged animal survival (100% of animal, from 22 to 32 and 33 days, respectively) and reduced the percentage of proliferating tumor cells detected by immunostaining of proliferation cell nuclear antigen (PCNA) (80 and 85% decrease, respectively). Moreover, the regulatory effects of these dietary n;-3 fatty acids on the influx of Ca(2+), activated by depletion of intracellular stores with thapsigargin (Tg), were investigated. By using a Ca(2+)-free/Ca(2+)-reintroduction protocol and Fura-2 as fluorescent indicator of intracellular free Ca(2+)([Ca(2+)](i)), we observed that EPA and DHA treatments markedly decreased Tg-induced rise in [Ca(2+)](i) (49 and 37% decrease, respectively). This effect was related to the inhibition of the store-operated Ca(2+) influx, as confirmed also by Mn(2+) influx experiments. The inhibitory action of EPA and DHA on the store-operated Ca(2+) influx could explain, at least in part, their antitumoral activity, as this Ca(2+) mobilization pathway appears to be involved in the cell signaling occurring in non-excitable cells to evoke many cellular processes, including cell proliferation.

Beta-carotene antagonizes the effects of eicosapentaenoic acid on cell growth and lipid peroxidation in WiDr adenocarcinoma cells.

The effects of combinations between eicosapentaenoic acid (EPA) and beta-carotene on cell growth and lipid peroxidation were investigated in human WiDr colon adenocarcinoma cells. EPA alone was able to inhibit the growth of WiDr cells in a dose- and time-dependent manner. Such an inhibition involved fatty acid peroxidation, as shown by the remarkable increase in the levels of Malondialdehyde (MDA) in EPA-treated cells. Beta-carotene was capable of reducing the growth inhibitory effects of EPA and the levels of MDA in a dose- and a time-dependent manner. In addition, EPA increased beta-carotene consumption in WiDr cells. This study provides evidence that beta-carotene can antagonize the effects of EPA on colon cancer cell growth and lipid peroxidation.

Eicosapentaenoic acid inhibits the growth of liver preneoplastic lesions and alters membrane phospholipid composition and peroxisomal beta-oxidation.

The purpose of this study was to determine whether individual administration of highly purified eicosapentaenoic acid (EPA), one of the main components of the n-3 polyunsaturated fatty acid family, would alter the growth of focal lesions during hepatocarcinogenesis. The protocol used to induce chemical carcinogenesis in liver was the Solt-Farber model (diethylnitrosamine as initiator and 2-acetylaminofluorene and carbon tetrachloride associated with partial hepatectomy as promoters). Proliferative lesions were quantified with the histochemical marker gamma-glutamyltranspeptidase at partial hepatectomy and at sacrifice. The number and size of the gamma-glutamyltranspeptidase-positive foci observed were significantly lower in rats supplemented with EPA. Fatty acid treatment increased EPA and docosapentaenoic acid content in membrane total phospholipids, in phosphatidylethanolamine, and in phosphatidylcholine. The content of arachidonic acid decreased significantly only in total phospholipids and in phosphatidylethanolamine. Fatty acid content of phosphatidylinositol was not modified. Moreover, we observed an increase in the activity of palmitoyl-CoA oxidase, the limiting enzyme of peroxisomal beta-oxidation, the preferential metabolic pathway of n-3 polyunsaturated fatty acid. Conversely, unmodified levels of alpha-tocopherol and unchanged production of lipid peroxidation products (malondialdehyde) were observed. These results suggest that the EPA inhibitory effect on preneoplastic foci development may be related to alteration of fatty acid composition in phospholipid classes and to enhancement of peroxisomal beta-oxidation and H2O2 production.

Cell proliferation, differentiation, and apoptosis are modified by n-3 polyunsaturated fatty acids in normal colonic mucosa.

Supplementation with low doses of eicosapentaenoic (EPA) or docosahexaenoic (DHA) acid was used here to investigate changes in epithelial proliferation, differentiation, and apoptosis in normal rat colonic mucosa. ACI/T rats received by oral administration low doses of purified EPA or DHA ethyl esters (1 g/kg body weight) and colonic mucosa was analyzed for cell proliferation, differentiation, and apoptosis. n-3 Polyunsaturated fatty acid incorporation into membrane phospholipids was investigated as reflections of fatty acid metabolism. Both EPA and DHA suppressed colonocyte proliferation and increased the numbers of differentiating and apoptotic cells without modification of the crypt morphology and the number of cells per crypt columns. A significant incorporation of the supplemented fatty acids into total phospholipids was observed. This enrichment was accompanied by a decreased content in arachidonic acid. The observation that EPA and DHA do not alter crypt morphology although they modify cell turnover in normal colonic mucosa suggests a possible use of these fatty acids as dietary chemopreventive agents.

Supplementation with canthaxanthin affects plasma and tissue distribution of alpha- and gamma-tocopherols in mice.

The effects of oral doses of canthaxanthin on tissue distribution of alpha- and gamma-tocopherols were investigated in three experiments in male and female Balb/c mice. Mice were assigned to receive canthaxanthin [7 or 14 microg/(g body weight.d)] or placebo (olive oil) by gavage for different periods of time (0, 1, 2, 4 and 6 wk). A 2 wk-treatment with canthaxanthin resulted in incorporation of the carotenoid in all tissues analyzed, including liver, spleen, kidney, lung and heart. In liver, the maximum accumulation of the carotenoid was reached after 2 wk of dosing in female mice and after 6 wk in male mice. Canthaxanthin incorporation was accompanied by changes in alpha- and gamma-tocopherol concentrations in plasma and tissues. These included the following: 1) a significant increase (P < 0.001) in alpha-tocopherol concentration in spleen (21 and 27% in male and female mice, respectively) after 2 wk and in liver ( approximately 50% in both male and female mice) after 6 wk; 2) a significant decrease in gamma-tocopherol concentration in plasma (P < 0.05) and tissues (P < 0.001) after 2 wk of treatment. In female mice, this decrease was 55% in plasma, 43% in liver, 44% in kidney, 71% in lung and 70% in heart. In male mice, the decrease was observed only in plasma (30%), kidney (54%) and heart (46%). In liver, the decrease in gamma-tocopherol concentration was both dose- and time-dependent and significantly (P < 0.001) greater in female than in male mice. We conclude that dietary administration of canthaxanthin modifies tocopherol status in murine tissues.