Jeffrey Atkinson

Pro-apoptotic Mechanisms of Action of a Novel Vitamin E Analog (α-TEA) and a Naturally Occurring Form of Vitamin E (δ-Tocotrienol) in MDA-MB-435 Human Breast Cancer Cells

Vitamin E derivative, RRR-α-tocopheryl succinate (vitamin E succinate, VES), is a potent pro-apoptotic agent, inducing apoptosis by restoring both transforming growth factor-β (TGF-β) and Fas (CD95) apoptotic signaling pathways that contribute to the activation of c-Jun N-terminal kinase (JNK)-mediated apoptosis. Objectives of these studies were to characterize signaling events involved in the pro-apoptotic actions of a naturally occurring form of vitamin E, δ-tocotrienol, and a novel vitamin E analog, α-tocopherol ether acetic acid analog [α-TEA; 2,5,7,8-tetramethyl-2R-(4R,8R,12-trimethyltridecyl)chroman-6-yloxyacetic acid]. Like VES, α-TEA and δ-tocotrienol induced estrogen-nonresponsive MDA-MB-435 and estrogen-responsive MCF-7 human breast cancer cells to undergo high levels of apoptosis in a concentration- and time-dependent fashion. Like VES, the two compounds induced either no or lower levels of apoptosis in normal human mammary epithelial cells and immortalized but nontumorigenic human MCF-10A cells. The pro-apoptotic mechanisms triggered by the structurally distinct α-TEA and δ-tocotrienol were identical to those previously reported for VES, that is, α-TEA- and δ-tocotrienol-induced apoptosis involved up-regulation of TGF-β receptor II expression and TGF-β-, Fas- and JNK-signaling pathways. These data provide a better understanding of the anticancer actions of a dietary form of vitamin E (δ-tocotrienol) and a novel nonhydrolyzable vitamin E analog (α-TEA).

The location and behavior of α-tocopherol in membranes

Vitamin E (alpha-tocopherol) has long been recognized as the major antioxidant in biological membranes, and yet many structurally related questions persist of how the vitamin functions. For example, the very low levels of alpha-tocopherol reported for whole cell extracts question how this molecule can successfully protect the comparatively enormous quantities of PUFA-containing phospholipids found in membranes that are highly susceptible to oxidative attack. The contemporary realization that membranes laterally segregate into regions of distinct lipid composition (domains), we propose, provides the answer. We hypothesize alpha-tocopherol partitions into domains that are enriched in polyunsaturated phospholipids, amplifying the concentration of the vitamin in the place where it is most needed. These highly disordered domains depleted in cholesterol are analogous, but organizationally antithetical, to the well-studied lipid rafts. We review here the ideas that led to our hypothesis. Experimental evidence in support of the formation of PUFA-rich domains in model membranes is presented, focusing upon docosahexaenoic acid that is the most unsaturated fatty acid commonly found. Physical methodologies are then described to elucidate the nature of the interaction of alpha-tocopherol with PUFA and to establish that the vitamin and PUFA-containing phospholipids co-localize in non-raft domains.

Differential Response of Human Ovarian Cancer Cells to Induction of Apoptosis by Vitamin E Succinate and Vitamin E Analogue, α-TEA

A vitamin E derivative, vitamin E succinate (VES; RRR-α-tocopheryl succinate), and a vitamin E analogue, 2,5,7,8-tetramethyl-2R-(4R,8R,12-trimethyltridecyl)chroman-6-yloxy acetic acid (α-TEA), induce human breast, prostate, colon, lung, cervical, and endometrial tumor cells in culture to undergo apoptosis but not normal human mammary epithelial cells, immortalized, nontumorigenic breast cells, or normal human prostate epithelial cells. Human ovarian and cervical cancer cell lines are exceptions, with α-TEA exhibiting greater proapoptotic effects. Although both VES and α-TEA can induce A2780 and subline A2780/cp70 ovarian cancer cells to undergo DNA synthesis arrest within 24 h of treatment, only α-TEA is an effective inducer of apoptosis. VES or α-TEA treatment of cp70 cells with 5, 10, or 20 μg/ml for 3 days induced 5, 6, and 19% versus 9, 36, and 71% apoptosis, respectively. Colony formation data provide additional evidence that cp70 cells are more sensitive to growth inhibition by α-TEA than VES. Differences in stability of the ester-linked succinate moiety of VES versus the ether-linked acetic acid moiety of α-TEA were demonstrated by high-performance liquid chromatography analyses that showed α-TEA to remain intact, whereas VES was hydrolyzed to the free phenol, RRR-α-tocopherol. Pretreatment of cp70 cells with bis-(p-nitrophenyl) phosphate, an esterase inhibitor, before VES treatment, resulted in increased levels of intact VES and apoptosis. Taken together, these data show α-TEA to be a potent and stable proapoptotic agent for human ovarian tumor cells and suggest that endogenous ovarian esterases can hydrolyze the succinate moiety of VES, yielding RRR-α-tocopherol, an ineffective apoptotic-inducing agent.

A mitochondria-targeted inhibitor of cytochrome c peroxidase mitigates radiation-induced death

The risk of radionuclide release in terrorist acts or exposure of healthy tissue during radiotherapy demand potent radioprotectants/radiomitigators. Ionizing radiation induces cell death by initiating the selective peroxidation of cardiolipin in mitochondria by the peroxidase activity of its complex with cytochrome c leading to release of hemoprotein into the cytosol and commitment to the apoptotic program. Here we design and synthesize mitochondria-targeted triphenylphosphonium-conjugated imidazole-substituted oleic and stearic acids which blocked peroxidase activity of cytochrome c/cardiolipin complex by specifically binding to its heme-iron. We show that both compounds inhibit pro-apoptotic oxidative events, suppress cyt c release, prevent cell death, and protect mice against lethal doses of irradiation. Significant radioprotective/radiomitigative effects of imidazole-substituted oleic acid are observed after pretreatment of mice from 1 hr before through 24 hrs after the irradiation.

Comparison of Vitamin E Derivatives α-TEA and VES in Reduction of Mouse Mammary Tumor Burden and Metastasis

A novel nonhydrolyzable ether derivative of RRR-α-tocopherol, RRR-α-tocopherol ether acetic acid analog [2, 5, 7, 8-tetramethyl-2R-(4R, 8R, 12-trimethyltridecyl)chroman-6-yloxyacetic acid (α-TEA)], and a hydrolyzable ester derivative RRR-α-tocopheryl succinate (vitamin E succinate; VES) inhibited BALB/c mouse 66cl-4-GFP mammary tumor cell growth in vitro and in vivo. Treatment of 66cl-4-GFP cells in culture with α-TEA or VES Induced dose-dependent DNA synthesis arrest and apoptosis and inhibited colony formation. Liposomal formulations of α-TEA delivered orally or by aerosol significantly reduced subcutaneous 66cl-4-GFP tumor burden and metastasis to lung and lymph nodes. Liposomal formulations of VES delivered by aerosol significantly reduced tumor burden and lung metastasis, but not lymph node metastasis. Unlike α-TEA, VES was ineffective in reducing tumor burden and metastasis to lungs and lymph nodes when administered orally. Analyses of tumor sections showed that α-TEA delivered by either method significantly reduced tumor cell proliferation as measured by KI67, and increased apoptosis as measured by terminal deoxynucleotldyl transferase-mediated dUTP-biotin nick-end labeling (TUNEL), whereas VES delivered by aerosol reduced tumor cell proliferation and increased apoptosis, but not significantly. In summary, the nonhydrolyzable ether vitamin E derivative α-TEA was effective in reducing tumor burden and metastasis when delivered either by aerosol or orally, whereas the hydrolyzable ester vitamin E derivative VES was effective only when delivered by aerosol.

Mechanisms of Ligand Transfer by the Hepatic Tocopherol Transfer Protein

α-Tocopherol is a member of the vitamin E family that functions as the principal fat-soluble antioxidant in vertebrates. Body-wide distribution of tocopherol is regulated by the hepatic α-tocopherol transfer protein (αTTP), which stimulates secretion of the vitamin from hepatocytes to circulating lipoproteins. This biological activity of αTTP is thought to stem from its ability to facilitate the transfer of vitamin E between membranes, but the mechanism by which the protein exerts this activity remains poorly understood. Using a fluorescence energy transfer methodology, we found that the rate of tocopherol transfer from lipid vesicles to αTTP increases with increasing αTTP concentration. This concentration dependence indicates that ligand transfer by αTTP involves direct protein-membrane interaction. In support of this notion, equilibrium analyses employing filtration, dual polarization interferometry, and tryptophan fluorescence demonstrated the presence of a stable αTTP-bilayer complex. The physical association of αTTP with membranes is markedly sensitive to the presence of vitamin E in the bilayer. Some naturally occurring mutations in αTTP that cause the hereditary disorder ataxia with vitamin E deficiency diminish the effect of tocopherol on the protein-membrane association, suggesting a possible mechanism for the accompanying pathology.

Besting Vitamin E: Sidechain Substitution is Key to the Reactivity of Naphthyridinol Antioxidants in Lipid Bilayers

A series of naphthyridinol analogs of α-tocopherol (α-TOH, right) with varying sidechain substitution was synthesized to determine how systematic changes in the lipophilicity of these potent antioxidants impact their radical-trapping activities in lipid bilayers, regenerability by water-soluble reductants, and binding to human tocopherol transport protein (TTP). The activities of the naphthyridinols were assayed in phosphatidylcholine unilamellar liposomes using a recently developed high-throughput assay that employs a boron dipyrromethene conjugate of α-TOH (H(2)B-PMHC) that undergoes fluorescence enhancement upon oxidation. The naphthyridinols afforded a dose-dependent protection of H(2)B-PMHC consistent with unprecedented peroxyl radical-trapping activity in lipid bilayers. While sidechain length and/or branching had no effect on their apparent reactivity, it dramatically impacted reaction stoichiometry, with more lipophilic compounds trapping two peroxyl radicals and more hydrophilic compounds trapping significantly less than one. It is suggested that the less lipophilic compounds autoxidize rapidly in the aqueous phase and that preferential partitioning of the more lipophilic compounds to the bilayer protects them from autoxidation. The cooperativity of a lipophilic naphthyridinol with water-soluble reducing agents was also studied in liposomes using H(2)B-PMHC and revealed superior regenerability by each of ascorbate, N-acetylcysteine, and urate when compared to α-TOH. Binding assays with human TTP, a key determinant of the bioavailability of the tocopherols, reveal that the naphthyiridinols can be very good ligands for the protein. In fact, naphthyridinols with sidechains of eight or more carbons had affinities for TTP which were similar to, and in one case 10-fold better than, α-TOH.

Preparation and biological assessment of hydroxycinnamic acid amides of polyamines

Many plants contain hydroxycinnamic acid conjugates of polyamines that are remarkably similar in general structure to the acylated polyamines found in spider and wasp toxins. In an effort to determine whether these compounds might play a role in the chemical defense of plants against arthropod pests we synthesized a variety of analogues of the coumaric (4-hydroxycinnamic) acid conjugates of di-, tri-, and tetraamines using common protection and acylation strategies. N(1)- and N(8)-coumaroyl spermidine were tested in feeding trials with insect larvae including the European corn borer (Ostrinia nubilalis), the tobacco budworm (Heliothis verescens) and the oblique banded leaf roller (Choristoneura rosaceana). Antifeedant assays with the rice weevil Sitophilus oryzae were also performed. Neither the naturally occurring coumaric acid conjugates of polyamines nor their analogues showed notable toxicity towards insects, despite precautions to maintain these easily oxidized materials in the wet diet. However, more direct bioassays of these compounds on glutamate dependent neuroreceptors including the deep abdominal extensor muscles of crayfish, or mammalian NMDA, delta2, and AMPA receptors, clearly showed that these compounds were inhibitory. N(1)-Coumaoryl spermine, a dodecyl and a cyclohexyl analogue were especially active at NMDA NR1/NR2B receptors. The latter had an IC(50) of 300 microM in the crayfish. N(1)-Coumaroyl spermine had an IC(50) in the crayfish preparation of 70-300 microM and against the mammalian NR1/NR2B receptor of 38 nM. Structure-activity variations show similar trends of length and hydrophobicity as has been seen previously with analogues of spider toxins. We conclude from this work that while the coumaric acid polyamine conjugates are active when directly applied to neuroreceptors, they show no overt toxicity when ingested by insect larvae.

Synthesis and Characterization of BODIPY-α-Tocopherol: A Fluorescent Form of Vitamin E

Fluorescent nitrobenzoxadiazole analogues of alpha-tocopherol (NBD-alpha-Tocs; lambda(ex) = 468 nm, lambda(em) = 527 nm) have been made previously to aid study of the intracellular location and transfer of vitamin E. However, these analogues are susceptible to photobleaching while under illumination for confocal microscopy as well as in in vitro FRET transfer assays. Here we report the synthesis of three fluorescent analogues of alpha-tocopherol incorporating the more robust dipyrrometheneboron difluoride (BODIPY) fluorophore. A BODIPY-linked chromanol should have no intervening polar functional groups that might interfere with binding to the hydrophobic binding site of the tocopherol transfer protein (alpha-TTP). A key step in bringing the two ring systems together was a metathesis reaction of vinyl chromanol and an alkenyl BODIPY. An o-tolyl containing second generation Grubbs catalyst was identified as the best catalyst for effecting the metathesis without detectable alkene isomerization, which when it occurred produced a mixture of chain lengths in the alkyl linker. C8-BODIPY-alpha-Toc 10c (lambda(ex) = 507 nm, lambda(em) = 511 nm, epsilon(507) = 83,000 M(-1) cm(-1)) having an eight-carbon chain between the chromanol and fluorophore, had the highest affinity for alpha-TTP (K(d) = 94 +/- 3 nM) and bound specifically as it could not be displaced with cholesterol.

Vitamin E isoforms directly bind PKCα and differentially regulate activation of PKCα

Vitamin E isoforms have opposing regulatory effects on leucocyte recruitment during inflammation. Furthermore, in vitro, vitamin E isoforms have opposing effects on leucocyte migration across endothelial cells by regulating VCAM (vascular cell-adhesion molecule)-1 activation of endothelial cell PKCα (protein kinase Cα). However, it is not known whether tocopherols directly regulate cofactor-dependent or oxidative activation of PKCα. We report in the present paper that cofactor-dependent activation of recombinant PKCα was increased by γ-tocopherol and was inhibited by α-tocopherol. Oxidative activation of PKCα was inhibited by α-tocopherol at a 10-fold lower concentration than γ-tocopherol. In binding studies, NBD (7-nitrobenz-2-oxa-1,3-diazole)-tagged α-tocopherol directly bound to full-length PKCα or the PKCα-C1a domain, but not PKCζ. NBD-tagged α-tocopherol binding to PKCα or the PKCα-C1a domain was blocked by diacylglycerol, α-tocopherol, γ-tocopherol and retinol, but not by cholesterol or PS (phosphatidylserine). Tocopherols enhanced PKCα-C2 domain binding to PS-containing lipid vesicles. In contrast, the PKCα-C2 domain did not bind to lipid vesicles containing tocopherol without PS. The PKCα-C1b domain did not bind to vesicles containing tocopherol and PS. In summary, α-tocopherol and γ-tocopherol bind the diacylglycerol-binding site on PKCα-C1a and can enhance PKCα-C2 binding to PS-containing vesicles. Thus the tocopherols can function as agonists or antagonists for differential regulation of PKCα.