Vitamin E protection against chemical-induced cell injury. II. Evidence for a threshold effect of cellular alpha-tocopherol in prevention of adriamycin toxicity

The cardiomyopathy produced by the widely used anticancer drug adriamycin (ADR) is believed to be related to the production of reaction oxygen species and consumption of reduced glutathione (GSH) during redox cycling of the drug. Protection by vitamin E against the toxicity of ADR was studied in a model of compromised isolated hepatocytes, generated by physiological alterations in the concentration of cell calcium. A decrease in cell calcium concentration leads to a greater loss of endogenous alpha-tocopherol and enhances the intracellular hydrolysis of exogenous alpha-tocopheryl esters. With this model, vitamin E (alpha-tocopheryl succinate) at 25 microM protected the calcium-depleted hepatocytes against the toxicity of ADR, in association with greater cellular alpha-tocopherol content as compared to calcium-adequate cells. The incubation of calcium-adequate hepatocytes with increasing concentrations of alpha-tocopheryl succinate up to 200 microM demonstrated that maximal protection by vitamin E was directly dependent on the alpha-tocopherol content of the cells, regardless of the concentration of cell calcium. The viability of the cells was closely associated with the alpha-tocopherol-mediated maintenance of cellular protein thiols. Viability and protein thiol content of the cells were maximal at cellular alpha-tocopherol levels in the range 0.6-1.0 nmol/10(6) cells in both calcium-depleted and -adequate cells. It is suggested that the potential use of vitamin E as a protective agent against ADR toxicity in vivo be reevaluated with an emphasis placed on the threshold level of intracellular alpha-tocopherol in the critical target tissue.     

Alpha-tocopheryl phosphate: a novel, natural form of vitamin E

We have detected alpha-tocopheryl phosphate in biological tissues including liver and adipose tissue, as well as in a variety of foods, suggesting a ubiquitous presence in animal and plant tissue. Alpha-tocopheryl phosphate is a water-soluble molecule that is resistant to both acid and alkaline hydrolysis, making it undetectable using standard assays for vitamin E. A new method was therefore developed to allow the extraction of both alpha-tocopheryl phosphate and alpha-tocopherol from a single specimen. We used ESMS to detect endogenous alpha-tocopheryl phosphate in biological samples that also contained alpha-tocopherol. Due to the significance of these findings, further proof was required to unequivocally demonstrate the presence of endogenous alpha-tocopheryl phosphate in biological samples. Four independent methods of analysis were examined: HPLC, LCMS, LCMS/MS, and GCMS. Alpha-tocopherol phosphate was identified in all instances by comparison between standard alpha-tocopheryl phosphate and extracts of biological tissues. The results show that alpha-tocopheryl phosphate is a natural form of vitamin E. The discovery of endogenous alpha-tocopheryl phosphate has implications for the expanding knowledge of the roles of alpha-tocopherol in biological systems.     

Alpha-tocopheryl succinate epitomizes a compound with a shift in biological activity due to pro-vitamin-to-vitamin conversion

With the advent of the third millennium, a number of pathologies have been eradicated or taken under control. However, the incidences, of cancer and atherosclerosis, the two most common causes of death in developed countries, have increased or, in some instances, only stagnated. Therefore there has been an intensive search for agents effective against such life-threatening conditions. Accordingly, the potential anti-atherogenic activity of vitamin E analogs has been studied extensively. Interestingly, recent reports strongly suggest that certain vitamin E analogs, represented in particular by alpha-tocopheryl succinate (alpha-TOS), also possess anti-neoplastic activity. In this communication, we review our current understanding of the molecular basis for these double effects of alpha-TOS and propose a testable hypothesis, according to which this semi-synthetic analog exerts both anti-atherogenic and anti-neoplastic activities. We propose that the prevalence of each activity depends on the actual form of the vitamin E analog. That is, the conversion of the pro-vitamin E form, alpha-TOS, to the corresponding vitamin form, alpha-tocopherol, makes this anti-neoplastic agent active against inflammatory diseases like atherosclerosis.     

Vitamin E protection against chemical-induced cell injury. I. Maintenance of cellular protein thiols as a cytoprotective mechanism

Vitamin E protection against chemical-induced toxicity to isolated hepatocytes was examined during an imbalance in the thiol redox system. Intracellular reduced glutathione (GSH) was depleted by two chemicals of distinct mechanisms of action: adriamycin, a cancer chemotherapeutic agent that undergoes redox cycling, producing reactive oxygen species that consume GSH, and ethacrynic acid, a direct depleter of GSH. The experimental system used both nonstressed vitamin E-adequate isolated rat hepatocytes and compromised hepatocytes subjected to physiologically induced stress, generated by incubation in calcium-free medium. At doses whereby intracellular GSH was near total depletion, cell injury induced by either chemical was found to follow the depletion of cellular alpha-tocopherol, regardless of the status of the GSH redox system. Changes in protein thiol contents of the cells closely paralleled the changes in alpha-tocopherol contents throughout the incubation period. Supplementation of the calcium-depleted hepatocytes with alpha-tocopheryl succinate (25 microM) markedly elevated their alpha-tocopherol content and prevented the toxicities of both drugs. The prevention of cell injury and the elevation in alpha-tocopherol contents were both associated with a prevention of the loss in cellular protein thiols in the near total absence of intracellular GSH. The mechanism of protection by vitamin E against chemical-induced toxicity to hepatocytes may therefore be an alpha-tocopherol-dependent maintenance of cellular protein thiols.     

Synthesis and study of the cancer cell growth inhibitory properties of alpha-, gamma-tocopheryl and gamma-tocotrienyl 2-phenylselenyl succinates

Vitamin E succinate selenium-conjugated molecules were synthesized and their apoptogenic properties were evaluated. 4-Methyl-2-phenylselenyl succinate (4) was prepared by the reaction of sodium benzeneselenolate with 2-bromosuccinic anhydrite in methanol solution. The methyl ester was converted to the acid (5) by hydrolysis with aqueous hydrochloric acid. Reaction of the 2-phenylselenyl succinic anhydrite (6) with alpha-tocopherol (1a), gamma-tocopherol (1c), and gamma-tocotrienol (2c) in acidic conditions gave the respective esters. The free radical scavenging properties of alpha-tocopheryl-2-phenylselenyl succinate (7), gamma-tocopheryl-2-phenylselenyl succinate (8), and gamma-tocotrienyl-2-phenylselenyl succinate (9) were evaluated in comparison with those of alpha-tocopheryl succinate (10), gamma-tocopheryl succinate (11), and gamma-tocotrienyl succinate (12), respectively, and the free tocopherols and gamma-tocotrienol. Compounds 7-9 induced a statistically significant decrease in prostate cancer cell viability compared to 10-12, respectively, or 5, exhibiting features of apoptotic cell death and associated with caspase-3 activation. These data show that structural modifications of vitamin E components by 5 enhance their apoptogenic properties in cancer cells.     

Vitamin E: action, metabolism and perspectives

Natural vitamin E includes four tocopherols and four tocotrienols. RRR-alpha-tocopherol is the most abundant form in nature and has the highest biological activity. Although vitamin E is the main lipid-soluble antioxidant in the body, not all its properties can be assigned to this action. As antioxidant, vitamin E acts in cell membranes where prevents the propagation of free radical reactions, although it has been also shown to have pro-oxidant activity. Non-radical oxidation products are formed by the reaction between alpha-tocopheryl radical and other free radicals, which are conjugated to glucuronic acid and excreted through the bile or urine. Vitamin E is transported in plasma lipoproteins. After its intestinal absorption vitamin E is packaged into chylomicrons, which along the lymphatic pathway are secreted into the systemic circulation. By the action of lipoprotein lipase (LPL), part of the tocopherols transported in chylomicrons are taken up by extrahepatic tissues, and the remnant chylomicrons transport the remaining tocopherols to the liver. Here, by the action of the "alpha-tocopherol transfer protein", a major proportion of alpha-tocopherol is incorporated into nascent very low density lipoproteins (VLDL), whereas the excess of alpha-tocopherol plus the other forms of vitamin E are excreted in bile. Once secreted into the circulation, VLDL are converted into IDL and LDL by the action of LPL, and the excess of surface components, including alpha-tocopherol, are transferred to HDL. Besides the LPL action, the delivery of alpha-tocopherol to tissues takes place by the uptake of lipoproteins by different tissues throughout their corresponding receptors. Although we have already a substantial information on the action, effects and metabolism of vitamin E, there are still several questions open. The most intriguing is its interaction with other antioxidants that may explain how foods containing small amounts of vitamin E provide greater benefits than larger doses of vitamin E alone.     

Interfacial properties of phosphatidylcholine bilayers containing vitamin E derivatives

alpha-Tocopherol and alpha-tocopheryl succinate are biologically active lipids. The activity of these lipids may be related to how they affect membrane physical-chemical properties. Utilizing fluorescence methods, we have investigated the effect of alpha-tocopherol, alpha-tocopheryl succinate, and alpha-tocopheryl acetate on the properties of model membranes consisting of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine and 1,2-dipalmitoyl-sn-glycero-3-phosphocholine. In liquid-crystalline phase phospholipid bilayers, alpha-tocopherol decreased acyl chain mobility and decreased the interfacial polarity, but had no effect on the interfacial surface charge. In contrast, alpha-tocopheryl succinate had little effect on acyl chain motion or interfacial hydration, but increased the interfacial surface charge. alpha-Tocopheryl acetate had very little effect on any of the measurements of these bilayer properties. In a gel phase bilayer, alpha-tocopherol decreased acyl chain order, whereas alpha-tocopheryl succinate and alpha-tocopheryl acetate did not. Each alpha-tocopheryl derivative had a different effect on interfacial polarity, however, only alpha-tocopheryl succinate increased the interfacial surface charge. The acylation of alpha-tocopherol abolishes its antioxidant activity and generates molecules with different membrane physical properties. The non-polar acetate group of alpha-tocopheryl acetate locates this compound in a region of the bilayer where it has little effect on bilayer interfacial properties. The free carboxyl group of alpha-tocopheryl succinate is located in the interfacial region of the bilayer where it increases the membrane surface charge.     

Vitamin E analogues as inducers of apoptosis: implications for their potential antineoplastic role

Recent evidence suggests that vitamin E and its analogues, which have been used for many years as antioxidants, may not only protect cells from free radical damage but also induce apoptotic cell death in various cell types. While alpha-tocopherol (alpha-TOH) is mainly known as an anti-apoptotic agent, its redox-silent analogues either have no influence on cell survival (alpha-tocopheryl acetate, alpha-TOA), or induce apoptosis (alpha-tocopheryl succinate, alpha-TOS). Although precise mechanisms of apoptosis induction by alpha-TOS remain to be elucidated, there is evidence that this process involves both the antiproliferative and membrane destabilising activities of the agent. Alpha-TOS has been shown to induce apoptosis in malignant cell lines but not, in general, in normal cells, and to inhibit tumorigenesis in vivo. These features suggest that this semi-synthetic analogue of vitamin E could be a promising antineoplastic agent.     

Hydrolysis of tocopheryl and retinyl esters by porcine carboxyl ester hydrolase is affected by their carboxylate moiety and bile acids

The objective of this study was to examine the in vitro hydrolysis of vitamin E esters (alpha-tocopheryl acetate, alpha-tocopheryl succinate and alpha-tocopheryl nicotinate) by pancreatic carboxyl ester hydrolase (CEH) at the concurrent presence of different bile acids at different concentrations. The assay was performed by measuring the amount of alpha-tocopherol released by porcine pancreatic juice upon addition to different solutions of alpha-tocopheryl esters, which were dispersed in bile acid mixed micelles at 37 degrees C, pH 7.4. The CEH activity was 10 U in the final assay, and the optimal concentration of cholate in this in vitro-system was determined to 30 mM for the hydrolysis of alpha-tocopheryl acetate. The hydrolysis of alpha-tocopheryl esters required presence of pancreatic juice and bile acids, and the results showed furthermore that the ability of pancreatic CEH towards hydrolysis of different alpha-tocopheryl esters increased with increasing lipophility, irrespective of the type or concentration of bile acid present in the assay. Likewise, retinyl palmitate was hydrolyzed at a faster rate than retinyl acetate. The structure of the bile acid influenced the rate of hydrolysis. Thus, cholate followed by glycodeoxy- and glycochenodeoxycholate were the most effective activators of CEH among the bile acids tested in this assay. The presence of gamma-tocopherol or all-trans-retinyl acetate in the assay showed a non-competitive inhibition of the hydrolysis rate of alpha-tocopheryl acetate.     

Relationship between cellular calcium and vitamin E metabolism during protection against cell injury

The extent of chemically induced injury to isolated hepatocytes has been previously shown to depend on the content of alpha-tocopherol in the cells, the levels of which are influenced by the concentration of extracellular calcium. Investigations into the effect of calcium on the alpha-tocopherol content of nonchemically exposed cells demonstrated that incubation of isolated hepatocytes in a calcium-deficient medium decreased cell calcium content to 10% of initial levels, and resulted in the depletion of endogenous alpha-tocopherol. This loss in alpha-tocopherol was not accounted for by alpha-tocopherylquinone formation. After supplementation of the cell incubation medium with alpha-tocopheryl succinate, the decreased cell calcium content was associated with higher levels of cellular alpha-tocopherol than in calcium-adequate cells. This was the result of greater intracellular hydrolysis of the tocopheryl ester in the calcium-depleted cells, and not an effect of extracellular calcium concentration on the uptake of alpha-tocopheryl succinate into the cells or on the extracellular hydrolysis of the ester. Uptake studies indicated a much greater achievable level of alpha-tocopherol in hepatocytes after incubation with alpha-tocopherol than with the alpha-tocopheryl ester. These data provide substantial support for the hypotheses that the content of extracellular calcium per se is not the determinant in toxic injury to hepatocytes, but that cell calcium content affects the intracellular metabolism of alpha-tocopherol and its esters, which may subsequently govern the outcome of a toxic challenge.     

Structural characteristic of terminal dicarboxylic moiety required for apoptogenic activity of alpha-tocopheryl esters

alpha-Tocopheryl succinate (TS) is known to induce apoptosis in various cells and has attracted attention as a chemotherapeutic agent. Recently, we reported the structural significance of the terminal dicarboxylic moiety for the action of TS [J. Nutr. Sci. Vitaminol. 49 (2003) 310-314]. In this study, to determine details of the relationship between the structure and the function of the terminal ester moiety of alpha-tocopherol (alpha-T), we synthesized four novel esters, alpha-tocopheryl oxalate (TO), alpha-tocopheryl malonate (TM), alpha-tocopheryl pimelate (TP) and alpha-tocopheryl succinate ethyl ester (TSE), and compared their apoptogenic activities with those of TS, alpha-T, gamma-tocopherol (gamma-T) and two commercially available alpha-T derivatives, alpha-tocopheryl nicotinate (TN) and alpha-tocopheryl acetate (TA), in vascular smooth muscle cells and a mouse breast cancer cell line C127I. TO and TM in addition to TS, but not the others, induced apoptosis in both cells. Particularly, TO was the most potent of all alpha-T derivatives used. The addition of exogenous superoxide dismutase (SOD) significantly prevented the apoptosis induced by TM as well as that by TS as reported previously, but did not affect TO-induced apoptosis. These results suggest that O(2)(-) generated exogenously participates in TM-induced apoptosis but not in TO-induced apoptosis. The difference in their apoptotic effects is attributed to structural properties of the terminal dicarboxylic moiety, which has an inflexible plane conformation in TO, while it is highly flexible in TM and TS.     

Influence of dietary vitamin E on the intermembrane transfer of alpha-tocopherol as mediated by an alpha-tocopherol binding protein

The effect of dietary vitamin E on the intermembrane transfer of (3R)-alpha-tocopherol, a spontaneous process accelerated in the presence of an alpha-tocopherol binding protein (alpha TBP), was examined. The transfer activity of this cytosolic liver protein was assayed via in vitro transfer of (3R)-alpha-[3H]tocopherol (alpha[3H]T) from egg lecithin liposomes to human erythrocyte ghosts (EG). Male Fisher 344 rats (1 and 20 months old) were fed diets containing 0, 30, and 500 mg/kg vitamin E (dl-alpha-tocopheryl acetate) for 15 weeks. Liver cytosol fractions were assayed for alpha[3H]T transfer activity (alpha TTA). Among young rats, those fed vitamin E-deficient diets had the highest alpha TTA, 5.02 +/- 3.10 pmole alpha[3H]T/min (mean +/- SD), which was different (P less than 0.05) from the spontaneous transfer rate of 2.10 pmole/min. Neither young rats fed 30 and 500 mg/kg vitamin E diets nor any of the aged rats showed alpha TTA which differed significantly from the spontaneous transfer rate. To examine the relationship between hepatic alpha-tocopherol levels and alpha TTA, alpha-tocopherol concentration per gram of wet liver was assayed by HPLC. A steep positive slope (6.39 +/- 1.46 pmole min-1 nmole g-1) and strong correlation (r = 0.873) between hepatic alpha-tocopherol and alpha TTA were observed (P less than 0.005) among young vitamin E-deficient rats. The data indicates that alpha TTA varies directly with hepatic alpha-tocopherol concentration when total liver vitamin E stores are very low. Thus, alpha TBP-mediated transfer of alpha-tocopherol may be manifest only when vitamin E status is compromised.     

Alpha-tocopherol is secreted from rat liver in very low density lipoproteins

Three separate studies were carried out to test the hypothesis that rat liver secretes vitamin E (alpha-tocopherol) within very low density lipoproteins (VLDL). i) When the clearance of plasma chylomicrons (CM) and VLDL was blocked by the administration of Triton WR-1339, alpha-tocopherol concentrations increased linearly with time in both classes of triacylglycerol-rich lipoproteins, although accumulation rates within VLDL exceeded those within CM. For fasted rats, appearance of alpha-tocopherol in VLDL persisted at slightly reduced rates. alpha-Tocopherol and triglycerides in the VLDL fraction responded to Triton WR-1339 administration by coordinate increases. In contrast to the situation in serum, alpha-tocopherol concentrations decreased in the liver following injection of Triton. ii) In order to inhibit the secretion of hepatic lipoproteins containing apolipoprotein B (apoB), rats were fed a diet containing orotic acid. This resulted in a reduction of apoB and alpha-tocopherol concentrations in serum and VLDL, whereas the vitamin E content of liver was increased. iii) In primary cultures of hepatocytes, alpha-tocopherol was secreted into the culture media predominantly within VLDL. We, therefore, conclude that the liver secretes alpha-tocopherol within VLDL and in this way contributes to the maintenance of serum vitamin E concentrations.     

Study on the specificity of alpha-tocopheryl (vitamin E) acid succinate effects on melanoma, glioma and neuroblastoma cells in culture

d- and dl-alpha-tocopheryl succinate inhibited growth and caused morphological changes in mouse melanoma (B-16), mouse neuroblastoma (NBP2), and rat glioma (C-6) cells in culture. To study whether the effects of alpha-tocopheryl (vitamin E) succinate on tumor cells are mediated by antioxidant mechanisms, the effects of lipid-soluble antioxidants, butylated hydroxyanisole (BHA) and butylated hydroxytoluene (BHT) were compared with those of vitamin E succinate. Results showed that these antioxidants produced alterations on the growth and morphology of neuroblastoma, melanoma, and glioma cells which are similar to those produced by vitamin E succinate; however, the extent of the effect depended upon the type of antioxidant and the form of tumor cells. These data suggest that the effects of vitamin E succinate on tumor cells may be mediated, in part, by antioxidant mechanisms.     

Enrichment of rat hepatic organelles by vitamin E administered subcutaneously

Novel modes of administering antioxidants to improve delivery to targeted tissues or cells may be advantageous in preventing oxidant-induced pathologies. Vitamin E (alpha-tocopherol) has been shown to be protective in several models of liver injury. The objectives of this study were: (1) to determine if subcutaneously (s.q.) administered emulsified vitamin E enriched liver and hepatic subcellular fractions with the antioxidant and (2) to carry out a time-dependent analysis of serum and tissue vitamin E in rats receiving daily s.q. vitamin E. In the first experiment rats injected daily s.q. with emulsified vitamin E for 9 d increased serum, total liver, liver mitochondria, and liver microsomes by 8-, 16-, 30-, and 29-fold, respectively, compared with placebo injections. Similar enrichment was observed after intramuscular injections. In the second experiment, daily doses of s.q. vitamin E increased liver concentrations 40-fold by 9 d, which decreased to 22-fold by 18 d, whereas serum adjusted vitamin E levels maximized with a 24-fold increase by day 3 and plateaued thereafter. In conclusion, s.q. administration of emulsified vitamin E to rats resulted in substantially elevated serum and liver concentrations of alpha-tocopherol compared with levels achievable by dietary supplementation. The s.q. route of administration is a potentially effective parenteral mode of delivery of vitamin E for conditions in which hepatic oxidative stress is present.     

Vitamin E bioavailability in humans

It is important to understand factors that can influence vitamin E bioavailability, particularly in populations with increased risk of coronary heart disease such as cigarette smokers. There is also a need to clarify the bioavailability of natural and synthetic vitamin E, which is currently a subject of some controversy. Previous studies using a competitive uptake approach have found bioavailability ratios of natural:synthetic vitamin E close to 2:1, differing from the accepted biopotency ratio of 1.36:1. We used a non-competitive uptake approach to compare the plasma biokinetics of deuterated natural (RRR) and synthetic (all rac) alpha-tocopheryl acetate in smokers and non-smokers. The study was comprised of two 4-week treatments with 400 mg/d (either RRR-alpha-tocopheryl or all rac-alpha-tocopheryl acetates), with a 12-week washout period between. Prior to and after each treatment subjects underwent a 48 h biokinetic protocol with 150 mg deuterated alpha-tocopheryl acetate in either the RRR or all rac form. Smokers had a lower deuterated alpha-tocopherol AUC than did non-smokers following administration of RRR, but there was no difference following administration of all rac. The ratio RRR:all rac from AUCs and C(max) was 1.3:1 in non-smokers and 0.9:1 in smokers. Following vitamin E supplementation, deuterated tocopherol AUCs were lower in both groups. These data suggest that non-smokers and smokers differ in their handling of vitamin E, that the relative bioavailability of natural and synthetic vitamin E is close to the currently accepted biopotency ratio of 1.36:1, and that following supplementation the ability of the plasma to take up newly absorbed vitamin E is decreased.     

Alpha-tocopherol modulates Cyp3a expression, increases gamma-CEHC production, and limits tissue gamma-tocopherol accumulation in mice fed high gamma-tocopherol diets

Although all forms of vitamin E are absorbed, the liver preferentially secretes alpha-, but not gamma-tocopherol, into plasma. Liver alpha-tocopherol secretion is under the control of the alpha-tocopherol transfer protein (TTP). Therefore, to assess gamma-tocopherol bioactivities Ttpa-/-, +/- and +/+ mice were fed for 5 weeks diets containing gamma-tocopherol 550 (gamma-T550), gamma-tocopherol 60 (gamma-T60) mg/kg that also contained trace amounts of alpha-tocopherol, a vitamin E-deficient diet, or a control diet. Plasma and tissues from mice fed gamma-T550 diets were found to contain similar gamma- and alpha-tocopherol concentrations despite the high dietary gamma-tocopherol content; nervous tissues contained almost no gamma-tocopherol. Liver vitamin E metabolites (carboxyethyl hydroxychromans, CEHCs) were also measured. In mice with widely ranging liver alpha- (from 0.7 to 16 nmol/g) and gamma-tocopherol concentrations (0 to 13 nmol/g), hepatic alpha-CEHC was undetectable, but gamma-CEHC concentrations (0.1 to 0.8 nmol/g) were correlated with both alpha- and gamma-tocopherol concentrations (P < 0.004). Hepatic cytochrome P450s (CYPs) involved in vitamin E metabolism, Cyp4f and Cyp3a, were also measured. There were no variations in Cyp4f protein expression as related to diet or mouse genotype. However, Cyp3a was correlated (P < 0.0001) with liver alpha-, but not gamma-tocopherol concentrations. These data support the hypothesis that alpha-tocopherol modulates xenobiotic metabolism by increasing Cyp3a expression, gamma-CEHC formation, and the excretion of both gamma-tocopherol and gamma-CEHC.     

alpha-Tocopheryl succinate induces apoptosis in prostate cancer cells in part through inhibition of Bcl-xL/Bcl-2 function

Although the antitumor effect of alpha-tocopheryl succinate (vitamin E succinate) has been well demonstrated, its underlying mechanism remains elusive. This study provides evidence that inhibition of Bcl-xL/Bcl-2 function represents a major pathway whereby alpha-tocopheryl succinate mediates apoptosis induction in prostate cancer cells. In vitro data indicate that alpha-tocopheryl succinate was able to disrupt the binding of Bak BH3 peptide to Bcl-xL and Bcl-2 with IC50 of 26 microm, in line with its potency in antiproliferation. Treatment of PC-3 cells with this agent led to reduced association of Bcl-2 and Bcl-xL with Bak, leading to caspase-dependent apoptosis. Moreover, overexpression of Bcl-xL protected LNCaP cells from the apoptosis induction. This mechanistic finding provided a basis to develop potent Bcl-xL/Bcl-2 inhibitors. Docking of alpha-tocopheryl succinate into the Bak peptide-binding site indicates that it adopted a unique hairpin-shaped conformation for protein interactions. We rationalized that the hemisuccinate and the two proximal isopranyl units of the side chain played a crucial role in ligand anchoring and protein-ligand complex stabilization, respectively. However, exposure of the distal isopranyl unit to a polar environment might diminish the binding affinity of alpha-tocopheryl succinate. This premise was corroborated by a structure-activity analysis of a series of derivatives with truncated side chains and/or altered carboxyl terminus. This computer model predicted that the removal of the distal isopranyl unit from the side chain would improve binding affinity, leading to two agents with significantly higher potency in inhibiting Bak peptide binding and in suppressing prostate cancer cell proliferation.     

Changes in n-6 and n-3 polyunsaturated fatty acids during lipid-peroxidation of mitochondria obtained from rat liver and several brain regions: effect of alpha-tocopherol

The effect of intraperitoneal administration of alpha-tocopherol (100 mg/kg weight/24 h) on ascorbate (0-0.4 mM) induced lipid peroxidation of mitochondria isolated from rat liver, cerebral hemispheres, brain stem and cerebellum was examined. The ascorbate induced light emission in hepatic mitochondria was nearly completely inhibited by alpha-tocopherol (control-group: 114.32+/-14.4; vitamin E-group: 17.45+/-2.84, c.p.m.x10(-4)). In brain mitochondria, 0.2 mM ascorbate produced the maximal chemiluminescence and significant differences among both groups were not observed. No significant differences in the chemiluminescence values between control and vitamin E treated groups were observed when the three brain regions were compared. The light emission produced by mitochondrial preparations was much higher in cerebral hemispheres than in brain stem and cerebellum. In liver and brain mitochondria from control group, the level of arachidonic acid (C20:4n6) and docosahexaenoic acid (C22:6n3) was profoundly affected. Docosahexaenoic in liver mitochondria from vitamin E group decreased by 30% upon treatment with ascorbic acid when compared with mitochondria lacking ascorbic acid. As a consequence of vitamin E treatment, a significant increase of C22:6n3 was detected in rat liver mitochondria (control-group: 6.42 +/-0.12; vitamin E-group: 10.52 +/-0.46). Ratios of the alpha-tocopherol concentrations in mitochondria from rats receiving vitamin E to those of control rats were as follows: liver, 7.79; cerebral hemispheres, 0.81; brain stem, 0.95; cerebellum, 1.05. In liver mitochondria, vitamin E shows a protector effect on oxidative damage. In addition, vitamin E concentration can be increased in hepatic but not in brain mitochondria. Lipid peroxidation mainly affected, arachidonic (C20:4n6) and docosahexaenoic (C22:6n3) acids.     

Membranotropic and pro-oxidative effects of alpha-tocopherol short-chain derivatives

The development of hemolysis and methemoglobin formation under influence of vitamin E derivatives was investigated on the in vitro model of rat red blood cells. It is established, that the shortening of lateral chains of alpha-tocopherol (T), alpha-tocopheryl acetate (TAc) and alpha-tocopheryl quinone (TQ) to 6 atoms of carbon (accordingly C6-T, C6-TAc, C6-TQ) results in occurrence of hemolysis and prooxidative properties, which are not characteristic of derivatives with native length of isoprenoid side chains. Modifications of chroman ring of short-chain derivatives, in turn, bring the contribution to mechanisms of their action. So, the effect of C6-TAc is caused, basically, physical destabilization of a plasmatic membrane, in C6-TQ effect prevails prooxidative mechanism, while C6-T equally shows membrane-destabilizing and prooxidative properties. Thus the derivatives T with the modified structure chroman nucleus and variability of isoprenoid side chains length show absolutely various properties and in each concrete case should be considered as independent biologically active substances irrespective of T properties.