Francesco Mazzini

Why tocotrienols work better: insights into the in vitro anti-cancer mechanism of vitamin E

The selective constraint of liver uptake and the sustained metabolism of tocotrienols (T3) demonstrate the need for a prompt detoxification of this class of lipophilic vitamers, and thus the potential for cytotoxic effects in hepatic and extra-hepatic tissues. Hypomethylated (γ and δ) forms of T3 show the highest in vitro and in vivo metabolism and are also the most potent natural xenobiotics of the entire vitamin E family of compounds. These stimulate a stress response with the induction of detoxification and antioxidant genes. Depending on the intensity of this response, these genes may confer cell protection or alternatively they stimulate a senescence-like phenotype with cell cycle inhibition or even mitochondrial toxicity and apoptosis. In cancer cells, the uptake rate and thus the cell content of these vitamers is again higher for the hypomethylated forms, and it is the critical factor that drives the dichotomy between protection and toxicity responses to different T3 forms and doses. These aspects suggest the potential for marked biological activity of hypomethylated “highly metabolized” T3 that may result in cytoprotection and cancer prevention or even chemotherapeutic effects. Cytotoxicity and metabolism of hypomethylated T3 have been extensively investigated in vitro using different cell model systems that will be discussed in this review paper as regard molecular mechanisms and possible relevance in cancer therapy.

Anticancer activity of vitamin E-derived compounds in murine C6 glioma cells.

for reproduction in rats, VE has many more important biological roles, such as the scavenging of reactive oxygen and nitrogen species, thus protecting the organism against the attack of free radicals, and the modulation of cellular signaling, enzymatic activity and gene expression in antioxidant and non-antioxidant manners. In recent years, evidence has accumulated on the antitumor and anti-inflammatory activity of VE metabolites and synthetic derivatives. The succinic monoester of a-tocopherol (1), atocopheryl succinate (aTOS, 2), a representative VE analogue, has been reported as a potent cytostatic and cytotoxic agent in several cancer cell models, showing high selectivity for malignant cells and low toxicity to normal cells. 11] It was proposed that the chroman ring of 2 is responsible for the activation of specific signaling pathways, such as PP2A/PKC, leading to cell-cycle arrest or apoptosis, while the presence of the succinyl ester further increases these biological responses leading to cell and mitochondria membrane destabilization. Furthermore, methylation of the terminal free carboxylic group leads to loss of proapoptotic activity. The limiting factor for the clinical application of 2, in particular regarding oral administration, is that the ester bond is prone to cleavage by esterases, releasing a-tocopherol, which is not active as an anticancer agent. In that regard, a-tocopheryloxyacetic acid (3, aTEA) is a promising VE analogue, which, unlike 2, has an acetic acid moiety attached to the chroman hydroxy group via a non-hydrolyzable ether bond. Compound 3 was shown to be very effective in terms of selectivity and potency in suppressing the growth of various tumors, both in vitro and in vivo. 14] Recently, other VE analogues with non-hydrolyzable bonds were reported with enhanced proapoptotic activity against Jurkat, U937 and human Meso-2 malignant mesothelioma cells compared to compound 2. 16] While structurally related to 2, the ester bond is replaced by an amide bond, the precursors being aand d-tocopheramine. Apart from the expected increased stability, a substantial improvement in the apoptotic activity is observed when going from the ester to the amide analogues and introducing an olefinic bond in the acid moiety. Those results suggest that the amide modification can be used as a starting point for the design of new VE analogues with potent antitumor activity. The molecular mechanism that causes the induction of apoptosis by VE analogues is still unclear. Monosuccinate 2 leads to destabilization of lysosomes and mitochondria by sphingomyelinase activation, ultimately targeting mitochondria through the generation of reactive oxygen species (ROS), affording the release of cytochrome c and the activation of proapoptotic proteins, such as caspase 9 and the Bcl-2 protein family. Apoptosis induced by the ether analogue 3 seems to proceed through another mechanism involving death receptor Fas signaling, the activation of JNK and its substrate c-Jun, truncation of Bid, conformational change of Bax, and activation of caspases 9 and 3. Our research involves the systematic study of the application of VE and related compounds in the treatment of glioblastoma—one of the most frequently occurring brain tumors, accounting for ~12–15 % of all brain tumors. Glioblastomas are among the most aggressive malignant human tumors, characterized by a diffuse local invasion of the normal parenchyma that renders complete surgical resection of cancerous tissue [a] Dr. F. Mazzini Dipartimento di Chimica e Chimica Industriale, Universit di Pisa Via Risorgimento 35, 56126 Pisa (Italy) Fax: (+ 39) 0502219260 E-mail : lcap@ns.dcci.unipi.it [b] Dr. M. Betti, B. Canonico, F. Luchetti, S. Papa Dipartimento di Scienze dell’Uomo, dell’Ambiente e della Natura, Universit degli Studi di Urbino “Carlo Bo”, Via C le Suore 2, 61029 Urbino (Italy) Fax: (+ 39) 0722304226 E-mail : michele.betti@uniurb.it [c] Dr. T. Netscher Research and Development, DSM Nutritional Products P.O. Box 2676, 4002 Basel (Switzerland) [d] Dr. F. Galli Dipartimento di Medicina Interna, Sez. di Biochimica Applicata e Scienze Nutrizionali, Universit di Perugia, Via del Giochetto, 06126 Perugia (Italy) Supporting information for this article is available on the WWW under http://dx.doi.org/10.1002/cmdc.200900492.

A first example of macromolecular Ti(IV) Lewis acid in the catalytic enantioselective Mukaiyama reaction

Abstract The preparation and characterization of a soluble copolymer P1 between styrene, divinylbenzene (DVB) and the enantiomerically pure salicylaldimine ligand 3c is described. The Lewis acid prepared from this macromolecular chiral ligand and Ti(OiPr)4 has been used in the catalytic Mukaiyama reaction to afford benzyl (R)-3-hydroxy-3-phenylpropanoate 7.

A comprehensive study for the validation of a LC–MS/MS method for the determination of free and total forms of urinary cortisol and its metabolites

Several pathological conditions can be related to the alteration of the urinary levels of cortisol (F) and its metabolites. The determination of each of them in the free and free plus conjugated form can provide a deeper insight into the impaired activity of the cortisol metabolism enzymes, thus improving the diagnosis protocol currently based only on the determination of total amount of urinary cortisol metabolites. In that view, an LC-MS/MS method for the determination of the free and total amount of urinary F, cortisone (E), tetrahydrocortisol (THF), allo-tetrahydrocortisol (A-THF) and tetrahydrocortisone (THE) was thus developed and validated. Deconjugation of glucocorticoids was carried out by enzymatic hydrolysis. Analytes were extracted by solid phase extraction, separated by liquid chromatography and analyzed via electro-spray ionization (negative ion mode) triple-quadrupole mass spectrometry in the selected reaction monitoring mode using a stable isotope-labeled internal standard. Baseline separation for all compounds, in particular the two stereoisomers A-THF and THF, was obtained. Matrix effects, not reported so far, were observed and minimized for the determination of urinary free E and THE. Validated range was 0.5-1000ng/mL for A-THF and THF, 5-800ng/mL for E and THE and 1-1000ng/mL for F, with R(2) values greater than 0.9981. The LOD and LOQ of the described method ranged from 0.1 to 3.0ng/mL, while the extraction recoveries resulted close to 100% for all the glucocorticoids determined. Precision and accuracy were well within ±10%. As suggested by the results obtained in the preliminary study on polycystic ovary syndrome (PCOS) urine samples, the method can be used to support clinical diagnosis of pathologies related to cortisol metabolism. In fact, levels of free and total glucocorticoids in control subjects were in agreement with previously reported data, as well as free and total A-THF/THF ratio in PCOS patients. Conversely, in the latter free F/E and A-THF+THF/THE ratios were lower than in control subjects (P<0.01), suggesting a possible alteration of 11β-HSD1 and 11β-HSD2 activity, to be further investigated.

Application of hyphenated mass spectrometry techniques for the analysis of urinary free glucocorticoids

Alteration of levels of glucocorticoids in plasma and urine can be related to several diseases. In particular, the determination of endogenous glucocorticoids in urine has been reported to provide information on cortisol and cortisone status, on the activities of steroid hormone enzymes and on glucocorticoid metabolism. In this study, the application of hyphenated mass spectrometry techniques (GC/MS without derivatization and LC/MS) for the simultaneous analysis of free urinary cortisol (F), cortisone (E), tetrahydrocortisol (THF), allo-tetrahydrocortisol (A-THF) and tetrahydrocortisone (THE) was evaluated. A sample preparation protocol by solid-phase extraction, mass spectrometry parameters and chromatographic conditions for both techniques were carefully optimized in terms of extracting phase and solvents, matrix effects, recovery, sensitivity and compound resolution. Baseline separation was achieved for the five underivatized analytes both in GC and LC. The LC/MS/MS technique was more suitable for the analysis of urine samples, being less influenced by matrix effects and showing excellent sensitivity and selectivity. A preliminary application of the reported method for the diagnosis of metabolic diseases was also described. The determination of each analyte in its free form, described for the first time in the paper, offers new perspectives in the application of glucocorticoid analysis for diagnostic purposes.

Circular dichroism of tocopherols versus tocotrienols.

Vitamin E is an essential nutrient of still increasing economic importance. Vitamin E derivatives include many nonracemic chiral compounds whose chirooptical characterization is scarcely described in the literature. We report the CD spectra of delta-tocopherol and its unsaturated analog delta-tocotrienol. TDDFT calculations demonstrate that the weak CD of delta-tocopherol is determined by the helicity of dihydropyrane ring. In addition, the moderate CD of delta-tocotrienol is due to the exciton interaction between the aromatic ring and the closest alkene group. Direct exciton-coupled CD calculations on structures generated by two different conformational sampling approaches reveal that, although such exciton coupling is expected to be weak, it is sufficient to explain the spectral differences between tocopherol and tocotrienol.

Mitochondrial-dependent anticancer activity of δ-tocotrienol and its synthetic derivatives in HER-2/neu overexpressing breast adenocarcinoma cells

Anticancer activity and mitochondrial mechanism of the vitamin E form δ-tocotrienol (δ-T3) was investigated in HER-2/neu-overexpressing human SKBR3 and murine TUBO breast cancer cells. δ-T3 was confirmed to possess high cytotoxic and apoptotic activity in SKBR3 cells as compared with all natural forms of vitamin E and several synthetic forms that included novel derivatives with the same backbone of δ-T3 such as δ-tocotrienyl-succinyl amide (δ-T3AS) and the redox-active analogue δ-tocotrienyl amine (δ-T3NH2). As observed in the case of alpha-TOS, a prototypical anticancer drug derived from α-tocopherol, succinylation of δ-T3 enhanced citotoxicity and apoptotic activity of the vitamer. δ-T3 induced apoptosis of SKBR3 cells was associated with mitochondrial destabilization, energy failure, and unbalanced activity of stress/survival MAPKs, namely p38 and ERK1/2 pathways. An increased generation of ROS followed to such a series of early events. Enhanced activity of δ-T3 in this human carcinoma cell line was characterized by the sustained uptake and oxidative transformation to the quinone derivative δ-T3Q, thereby suggesting redox effects in SKBR3 mitochondria by this vitamer. Viability and uptake data show a different pattern of responses in TUBO cells with higher response to synthetic derivatives of δ-T3 than in SKBR3 cells. In conclusion, synthetic derivatives of δ-T3 with enhanced apoptotic activity in breast carcinoma cells are investigated for the first time in this study also describing mechanistic aspects of mitochondrial effects of δ-T3. Further investigation in preclinical models of HER2/neu-high breast adenocarcinoma is underway to identify other and more effective forms of VE in this type of cancer.

Analytical strategies to assess the functional metabolome of vitamin E.

After more than 90 years from its discovery and thousands of papers published, the physiological roles of vitamin E (tocopherols and tocotrienols) are still not fully clarified. In the last few decades, the enzymatic metabolism of this vitamin has represented a stimulating subject of research. Its elucidation has opened up new horizons to the interpretation of the biological function of that class of molecules. The identification of specific properties for some of the physiological metabolites and the definition of advanced analytical techniques to assess the human metabolome of this vitamin in vivo, have paved the way to a series of hypotheses on the functional implications that this metabolism may have far beyond its catabolic role. The present review collects the available information on the most relevant analytical strategies employed to assess the status and metabolism of vitamin E in humans as well as in other model systems. A particular focus is dedicated to the analytical methods used to measure vitamin E metabolites, and particularly long-chain metabolites, in biological fluids and tissues. Preliminary information on a new LC-APCI-MS/MS method to measure these metabolites in human serum is reported.

Immobilization of matrix metalloproteinase 8 (MMP-8) for online drug screening

Matrix metalloproteinase 8 (MMP-8) has been reported to have a key role in several pathologic conditions, like heart diseases, osteoarthritis, multiple sclerosis, and various other inflammatory conditions. Therefore, there is a great interest regarding the development of MMP-8 selective inhibitors. In the recent years, immobilized enzyme reactors (IMERs) proved to be an efficient alternative to solution-based assays. Besides the recycling of the enzyme, IMER approach allows a simple way to determine affinity data and thus the ranking of inhibiting potency of the compounds under study, especially when coupled to MS. In this study, the immobilization of MMP-8 was investigated in terms of type of support, kinetic parameters, storage and pH stability. Epoxy activated silica resulted the best matrix for the preparation of an immobilized enzyme reactor (IMER) containing human MMP-8. The IMER was successfully used for the online screening of known MMP-8 inhibitors in zonal chromatography and inhibition experiments.

Tocopheramines and tocotrienamines as antioxidants: ESR spectroscopy, rapid kinetics and DFT calculations

Tocopheramines (TNH2) and tocotrienamines (T3NH2) are analogues of tocopherols (TOH) and tocotrienols in which phenolic OH is replaced by NH2. It was shown in previous studies that TNH2 and T3NH2 act as potent antioxidants. In this study we compared the one-electron oxidation of TNH2/T3NH2 by diphenyl picryl hydrazyl (DPPH) and galvinoxyl (GOX) radicals with the one of α-TOH as a reference compound using ESR spectroscopy, stopped flow spectrophotometry and density functional theory (DFT) calculations. ESR spectroscopy revealed the presence of tocopheramine radicals during electrochemical oxidation of α-TNH2. Kinetic measurements demonstrated that in apolar n-hexane TNH2/T3NH2 derivatives reacted two to three orders of magnitude slower than α-TOH with the model radicals. DFT calculations indicated that this correlates well with the higher bond dissociation energy (BDE) for N-H in TNH2 than for O-H in α-TOH in pure H-atom transfer (HAT). In the more polar medium ethanol TNH2/T3NH2 derivatives partially reacted faster than α-TOH depending on the reaction partner. DFT calculations suggest that this is due to reaction mechanisms alternative to HAT. According to thermochemistry data sequential proton loss and electron transfer (SPLET) is more favored for α-TOH in ethanol than for TNH2. Therefore, for TNH2 a contribution of the alternative mechanism of sequential electron transfer-proton transfer (SET-PT) could be a possible explanation. These data show that the antioxidant reactivity strongly depends on the structure, reaction partners and environment. According to these findings TNH2/T3NH2 should be superior as antioxidants over α-TOH in polar head group regions of membranes but not in the apolar core of lipid bilayers.