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.

Differential effects of sphingomyelin hydrolysis and resynthesis on the activation of NF-kappa B in normal and SV40-transformed human fibroblasts.

The precise role of ceramide in NF-kappaB signaling remains unclear. The recent observation of differential sphingomyelin synthase (SMS) activity in normal (low SMS) versus SV40-transformed (high SMS) WI38 human lung fibroblasts provides an opportunity to assess the involvement of ceramide and SMS in NF-kappaB activation. Treatment of normal WI38 fibroblasts with bacterial sphingomyelinase resulted in a 4-fold elevation of ceramide and blocked NF-kappaB activation by serum stimulation. Such inhibition was not observed in SV40-transformed fibroblasts. Under regular growth conditions, after sphingomyelinase was washed out, normal WI38 did not show SM re-synthesis nor NF-kappaB activation. In SV40-WI38, on the other hand, sphingomyelinase washout induced resynthesis of SM due to the action of SMS on ceramide generated at the plasma membrane. NF-kappaB activation correlated with SM resynthesis. This activation was abrogated by D609, which inhibited SM resynthesis but not the initial formation of ceramide. The differential activity of SMS may explain the effects of ceramide in NF-kappaB signaling: in the absence of significant SMS activity, ceramide inhibits NF-kappaB, whereas with high SMS, the conversion of the ceramide signal to a diacylglycerol signal by the action of SMS stimulates NF-kappaB. These results also suggest a role for SMS in regulating NF-kappaB.

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.

Canthaxanthin induces apoptosis in human cancer cell lines.

To investigate the possibility that canthaxanthin inhibits cancer cell growth by inducing apoptosis, human WiDr colon adenocarcinoma and SK-MEL-2 melanoma cells were treated with two different doses of the carotenoid for 48 h. Canthaxanthin was incorporated and/or associated to cells. The treatment with the carotenoid caused growth inhibition in both cell types. Concomitantly, apoptosis was induced. Increasing time of exposure and carotenoid concentration, this effect was more pronounced. At 48 h, the percentages of apoptotic cells were 13 and 15, using 1 microM canthaxanthin, and 18 and 20, using 10 microM canthaxanthin in WiDr and SK-MEL-2 cells, respectively. This study represents the first demonstration that canthaxanthin is able to induce apoptosis in tumour cells.

Dietary supplementation with eicosapentaenoic and docosahexaenoic acid inhibits growth of Morris hepatocarcinoma 3924A in rats: effects on proliferation and apoptosis.

The effect of individual administration of low doses of highly purified eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) (1 g/kg body weight) on the growth of Morris hepatocarcinoma 3924A transplanted in ACI/T rats was investigated. Both EPA and DHA inhibited growth of the hepatocarcinoma (50% reduction of tumor weight or volume at the 19th day after transplantation for both of the n-3 PUFA groups). EPA treatment reduced the percentage of proliferating tumor cells labeled with BUdR (10-fold), whereas DHA did not. Conversely, DHA supplementation induced a doubling of the number of cells undergoing apoptosis (labeled by TUNEL), whereas EPA treatment was much less effective. Analysis of changes in phospholipid fatty acids in tumor-cell membranes after both treatments with EPA and DHA showed a significant reduction in arachidonic-acid levels. EPA and docosapentaenoic acid (DPA), its elongation product, were increased in the phospholipids from EPA-treated animals. DHA and EPA, but not DPA, were increased in the DHA-treated group. It is concluded from the results of the present study that the anti-tumoral effect of EPA is related mainly to its inhibition of cell proliferation, whereas that of DHA corresponds with its induction of apoptosis. The alterations in fatty-acid composition induced by EPA or DHA appear to be factors underlying their differential actions on cell proliferation and apoptosis.

Low-dose eicosapentaenoic or docosahexaenoic acid administration modifies fatty acid composition and does not affect susceptibility to oxidative stress in rat erythrocytes and tissues.

In view of the promising future for use of n-3 polyunsaturated fatty acids (PUFA) in the prevention of cancer and cardiovascular diseases, it is necessary to ensure that their consumption does not result in detrimental oxidative effects. The aim of the present work was to test a hypothesis that low doses of eicosapentaenoic acid (EPA) or docosahexaenoic acid (DHA) do not induce harmful modifications of oxidative cell metabolism, as modifications of membrane fatty acid composition occur. Wistar rats received by gavage oleic acid, EPA, or DHA (360 mg/kg body weight/day) for a period of 1 or 4 wk. Fatty acid composition and alpha-tocopherol content were determined for plasma, red blood cell (RBC) membranes, and liver, kidney, lung, and heart microsomal membranes. Susceptibility to oxidative stress induced by tert-butylhydroperoxide was measured in RBC. EPA treatment increased EPA and docosapentaenoic acid (DPA) content in plasma and in all the membranes studied. DHA treatment mainly increased DHA content. Both treatments decreased arachidonic acid content and n-6/n-3 PUFA ratio in the membranes, without modifying the Unsaturation Index. No changes in tissue alpha-tocopherol content and in RBC susceptibility to oxidative stress were induced by either EPA or DHA treatment. The data suggest that EPA and DHA treatments can substantially modify membrane fatty acids, without increasing susceptibility to oxidative stress, when administered at low doses. This opens the possibility for use of low doses of n-3 PUFA for chemoprevention without risk of detrimental secondary effects.

Antioxidant and prooxidant role of beta-carotene in murine normal and tumor thymocytes: effects of oxygen partial pressure.

The effects of the partial pressure of oxygen (pO2) on antioxidant efficiency of beta-carotene in inhibiting radical-initiated lipid peroxidation were studied in murine normal and tumor thymocytes. At 150 mm Hg pO2 (the pressure of oxygen in normal air), beta-carotene acted as an antioxidant, inhibiting radical-induced lipid peroxidation in both normal and tumor thymocytes. At 760 mm Hg p02, beta-carotene lost its antioxidant activity in normal thymocytes and exhibited a dose-dependent prooxidant effect in tumor thymocytes. In these cells, the prooxidant effect of beta-carotene was also accompanied by an increase of endogenous alpha-tocopherol loss. beta-Carotene radical-trapping and autooxidation reactions were faster at 760 mm Hg pO2 than at 150 mm Hg pO2 in both normal and tumor thymocytes and the carotenoid was more rapidly consumed in tumor cells. These data point out a key role of the oxygen tension on the antioxidant effectiveness of beta-carotene. They also show a selective prooxidant effect of beta-carotene under 100% oxygen in tumor cells.

Antitumor effect of an oral administration of canthaxanthin on BALB/c mice bearing thymoma cells.

The antitumor effect of canthaxanthin in BALB/c mice bearing a transplantable thymoma was investigated. Male or female mice received two different doses of canthaxanthin (7 or 14 micrograms/g body wt/day) starting 15 days before tumor inoculation (7 x 10(7) cells i.p.). Canthaxanthin treatment delayed the appearance of macroscopic ascites and prolonged animal survival. This effect was dose dependent and more evident in females than in males. It appeared only when the carotenoid was administered before tumor transplantation. The antitumor efficacy of the carotenoid was related to its tissue incorporation. Canthaxanthin was incorporated in a dose-dependent manner in liver and thymoma cells and to a larger extent in females than in males. Our study shows the antitumor efficacy of canthaxanthin in vivo against a transplantable murine thymoma and points out the importance of dose, administration timing, and sex in the antitumor efficacy of this compound.

n-3 fatty acids induce oxidative modifications in human erythrocytes depending on dose and duration of dietary supplementation.

The present work was performed to study an optimal dose and duration of dietary n-3 polyunsaturated fatty acid (PUFA) supplementation that would not result in harmful modifications of oxidative cell metabolism. Forty healthy subjects were divided into four groups that received 2.5 g/d eicosapentaenoic acid (EPA) + docosahexaenoic acid (DHA), 5.1 g EPA + DHA/d, 7.7 g EPA + DHA/d, or placebo. Fatty acid composition, tocopherol status, and susceptibility to lipid peroxidation induced in vitro by 2,2'-azobis-(2-amidinopropane) (AAPH) were evaluated in human red blood cell (RBC) membranes on days 30 and 180. n-3 PUFA treatment increased EPA and DHA concentrations in RBC membranes in a time-dependent manner in all of the n-3 PUFA groups. These modifications occurred with concomitant dose- and time-dependent increases in the membrane unsaturation index. After 30 d of treatment with n-3 PUFAs, alpha-to-copherol significantly increased in RBC membranes of the intermediate- and high-dose groups. Because of the higher concentration of this antioxidant in these groups, the susceptibility of RBC membranes to peroxidation was decreased. However, after 180 d of treatment, alpha-tocopherol decreased to baseline values and AAPH-induced lipid peroxidation increased in a dose-dependent manner. These results show that high doses of dietary n-3 PUFAs, as well as long-time treatments, affect human RBC susceptibility to lipid peroxidation by changes in fatty acid composition and tocopherol content.

Effect of beta-carotene and canthaxanthin on tert-butyl hydroperoxide-induced lipid peroxidation in murine normal and tumor thymocytes.

It has been reported that the anticarcinogenic effect of carotenoids could be related to an antioxidant mechanism. The antioxidant efficiency of beta-carotene and canthaxanthin was evaluated in murine normal and tumor thymocytes. Normal and tumor cells were exposed under air to tert-butyl hydroperoxide (t-BOOH) and lipid peroxidation was measured in the absence or in the presence of the two carotenoids. Our results show that: (a) Both carotenoids, added at effective and comparable concentrations (from 1 to 50 microM), were able to inhibit t-BOOH-induced malondialdehyde formation in a dose-dependent manner. (b) Canthaxanthin was a more potent antioxidant that beta-carotene. (c) The inhibition of lipid peroxidation was greater in tumor thymocytes. (d) Carotenoids were consumed differentially during the incubation with the prooxidant. beta-Carotene was consumed faster than canthaxanthin and in a larger amount in tumor than in normal thymocytes. The addition of the iron chelator deferoxamine or the SH group reducing agent dithiothreitol reduced t-BOOH-induced beta-carotene consumption in tumor cells but not in normal ones. (e) The loss of endogeneous alpha-tocopherol induced by t-BOOH was enhanced by the addition of beta-carotene, suggesting the possibility of oxidative interactions between the two antioxidants. These results confirmed the antioxidant effectiveness of carotenoids in normal and tumor cells, although differences depending on the kind of cells and carotenoids used were found.

d-alpha-tocopherol inhibition of vascular smooth muscle cell proliferation occurs at physiological concentrations, correlates with protein kinase C inhibition, and is independent of its antioxidant properties.

d-alpha-Tocopherol, but not d-beta-tocopherol, negatively regulates proliferation of vascular smooth muscle cells at physiological concentrations. d-alpha-Tocopherol inhibits protein kinase C (PKC) activity, whereas d-beta-tocopherol is ineffective. Furthermore d-beta-tocopherol prevents the inhibition of cell growth and of PKC activity caused by d-alpha-tocopherol. The negative regulation by d-alpha-tocopherol of PKC activity appears to be the cause and not the effect of smooth muscle cell growth inhibition. d-alpha-Tocopherol does not act by binding to PKC directly but presumably by preventing PKC activation. It is concluded that, in vascular smooth muscle cells, d-alpha-tocopherol acts specifically through a nonantioxidant mechanism and exerts a negative control on a signal transduction pathway regulating cell proliferation.

Prooxidant activity of beta-carotene under 100% oxygen pressure in rat liver microsomes.

The effects of the partial pressure of oxygen (pO2) on antioxidant efficiency of beta-carotene in inhibiting 2,2'-azobis(2-amindinopropane) (AAPH)-induced lipid peroxidation are investigated in rat liver microsomal membranes. The rate of peroxyl radicals generated by thermolysis of AAPH at 37 degrees C is markedly higher at 150 than 760 mm Hg pO2. At 150 mm Hg Po2 beta-carotene acts as an antioxidant, inhibiting 2,2'-azobis(2-amidinopropane) 9AAPH0-induced Malonidaldehyde (MDA) formation. At 760 mm Hg Po2, it loses its antioxidant activity and shows a prooxidant effect, increasing lipid peroxidation products. alpha-Tocopherol prevents the prooxidant effect of beta-carotene in a dose-dependent manner. Our data provide the first evidence of a prooxidant effect of beta-carotene under 100% oxygen pressure in a biological membrane model and point out the existence of cooperative interactions between beta-carotene and alpha-tocopherol.

Dietary fish oil inhibits human erythrocyte Mg,NaK-ATPase.

The effects of long-term treatment with a high dose (7.7 g/day) of n-3 polyunsaturated fatty acids (PUFA) were studied for human red blood cells (RBCs). RBCs isolated from healthy subjects treated for 30 and 180 days with n-3 PUFA showed the following modifications: (1) a time dependent modification of membrane fatty acid composition with a concomitant increase in membrane lipid unsaturation; (2) an increase in lipid peroxidation, expressed as malondialdehyde release, induced in vitro by t-butyl hydroperoxide (t-BOOH); (3) a time-dependent decrease in susceptibility to hemolysis, expressed as K+ leakage, induced in vitro by t-BOOH; (4) a time-dependent decrease in total and ouabain-insensitive Mg,NaK-ATPase activity. These results suggest that long term dietary supplementation with high doses of n-3 PUFA significantly modifies RBC structure and function that might lead to harmful side effects.

The effect of fatty acid unsaturation on the antioxidant activity of beta-carotene and alpha-tocopherol in hexane solutions.

The effect of fatty acid unsaturation on the antioxidant ability of beta-carotene and alpha-tocopherol to inhibit azobis-isobutyronitrile AIBN)-induced malondialdehyde (MDA) formation is investigated in a hexane solution. A positive correlation is shown between the fatty acid unsaturation and MDA production in homogeneous solutions. Both beta-carotene and alpha-tocopherol act as chain-breaking antioxidants in our model, effectively suppressing AIBN-induced MDA formation. When alpha-tocopherol is added to fatty acid solutions, a lag phase of about 30 min is observed and a propagation phase is produced at a rate dependent on the degree of unsaturation and similar to that observed in the absence of the antioxidant. A specific inhibition of initiation phase by alpha-tocopherol is confirmed by its total consumption after 30 min of incubation with AIBN. On the other hand, when beta-carotene is added, a lag period is not observed and the inhibition of propagation phase progressively increases in relation to the degree of fatty acid unsaturation. These data present different antioxidant roles for beta-carotene and alpha-tocopherol in AIBN-induced lipid peroxidation and suggest that beta-carotene can be a very effective antioxidant in highly unsaturated membranes, such as those enriched with n-3 polyunsaturated fatty acids (PUFA).