The suppression of ornithine decarboxylase expression and cell proliferation at the promotion stage of lung tumorigenesis in mice by alpha-tocopheryloxybutyric acid.

It is known that vitamin E inhibits tumor cell growth in vitro irrespective of its antioxidative effect. However, it is unclear whether the effect in vitro can be applied to the in vivo situation. In order to address this question, we estimated if alpha-tocopheryloxybutyric acid (TSE), a non-antioxidative vitamin E derivative in vivo, could inhibit cell proliferation during the tumorigenic process of lung in mice treated with 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK), the most potent carcinogen among tobacco-specific nitrosamines. TSE administration suppressed the labeling index of the proliferating cell nuclear antigen, a marker of cell proliferation at a promotion phase of NNK-induced lung tumorigenesis in mice. Similarly, TSE administration inhibited the elevation of ornithine decarboxylase (ODC) activity and its mRNA at the promotion phase. Of four transcription factors contributing to ODC induction, the change in the level of the c-Myc/Max-consensus oligonucleotide complex was only proportional to the change in ODC mRNA level. These results suggest that vitamin E can inhibit cell proliferation linked with ODC induction at the promotion phase of lung tumorigenesis irrespective of its antioxidative effect and that modulation of the transactivation of the c-Myc/Max complex for the ODC gene by TSE in part contributes to the suppression of ODC induction.

Vitamin E inhibits cell proliferation and the activation of extracellular signal-regulated kinase during the promotion phase of lung tumorigenesis irrespective of antioxidative effect.

We have already reported that the activation of extracellular signal-regulated kinase (Erk) is critical in the stimulation of cell proliferation during the promotion stage of urethane-induced lung tumorigenesis in mice. Also, we have found that vitamin E suppresses lung tumorigenesis by inhibiting cell proliferation at the promotion stage. However, it is still unclear whether this inhibitory effect at the promotion stage is based on the antioxidative effect of vitamin E or not. In order to address this question, we examined the inhibitory effect of alpha-tocopheryloxybutyric acid (TSE), an ether derivative of vitamin E that cannot act as an antioxidant in vivo, on cell proliferation and the activation of Erk during promotion of lung tumorigenesis. On day 30 after urethane injection (750 mg/kg, i. p.) in A/J mice, TSE or vitamin E at 100 micromol/kg, p.o. was administered. Twenty-four hours after the final administration, the mice were killed to analyze cell proliferation and related parameters. The labeling index of proliferating cell nuclear antigen (a marker of cell proliferation) and ornithine decarboxylase activity (a marker of the promotion stage in lungs) were attenuated by treatment with TSE or vitamin E. TSE or vitamin E treatment also inhibited urethane-induced activation of Erk and suppressed the activation of other essential members of the Erk cascade (Ras, Raf and Mek). These results suggest that vitamin E inhibits cell proliferation and activation of the Erk cascade during promotion of urethane-induced lung tumorigenesis in mice, independent of its antioxidative effect.

Michael-type reaction of ethyl bromodifluoroacetate with alpha,beta- unsaturated carbonyl compounds in the presence of copper powder.

We have reported that the reaction of ethyl bromodifluoroacetate (1) with alkenyl iodides in the presence of copper powder gives ethyl alkenyldifluoroacetates. As an extension of this reaction, reaction of 1 with Michael acceptors in the presence of copper powder was examined and found to give 1,4-addition products selectively, unless the acceptor has a group stabilizing a radical intermediate, such as a phenyl group.

Synthesis of new synthons for organofluorine compounds from halothane containing sulfur functional groups.

To develop new synthons for the syntheses of organofluorine compounds, the treatment of Halothane, 2-bromo-2-chloro-1,1,1-trifluoroethane, (1) with 4-methylbenzenethiol (2) in the presence of sodium hydride gave 1-chloro-2,2,2-trifluoroethyl 4-methylphenyl sulfide (3), which was oxidized with m-chloroperbenzoic acid (m-CPBA) to the corresponding sulfoxide (4) and sulfone (5). Reaction of 3 and 5 with allyltributyltin in the presence of 2,2'-azobis(isobutyronitrile) (AIBN) gave 1-(trifluoromethyl)-3-butenyl compounds (9, 11). Sulfoxide 4 was decomposed in this condition. The treatment of 3 with allyltrimethylsilane in the presence of Lewis acids gave 1-(trifluoromethyl)-3-butenyl compounds (9) in good yield. This result suggests that 4-methylphenylthio substituent stabilizes the alpha-carbocation effectively, though the trifluoromethyl group destabilizes it strongly. Aromatic compounds similarly reacted with 3 in the presence of titanium(IV) chloride to give 2-aryl-1,1,1-trifluoro-2-(4-methylphenylthio)ethanes. Thus, sulfur compounds derived from Halothane were found to be useful new synthons for organofluorine compounds.

Synthesis of fluorine analogs of protoporphyrin potentially useful for diagnosis and therapy of cancer. IV. Synthesis of (trifluorovinyl)vinyl- and (1-chloro-2,2-difluorovinyl)vinyldeuteroporphyrins.

Trifluoro or chlorodifluoro analogs of protoporphyrin, the compounds in the title, were synthesized for use in the diagnosis and therapy of cancer. 3- Or 8-acetyldeuteroporphyrin dimethyl esters (2 and 3) were iodinated with iodine in the presence of potassium carbonate to the corresponding iodo compounds (5 and 6). The iodo compounds (5 and 6) were treated with bis(trifluorovinyl)zinc in the presence of tetrakis(triphenylphosphine)-palladium to give trifluorovinyl derivatives (7 and 8) in good yields. Reduction of the acetyl group of 7 and 8 with sodium borohydride afforded the corresponding hydroxyethyl derivatives (9 and 10). Compounds (9 and 10) were dehydrated with methanesulfonyl chloride and triethylamine to give (trifluorovinyl)vinyldeuteroporphyrin dimethyl esters (11 and 12). Treatment of 5 and 6 with bis(1-chloro-2,2-difluorovinyl)zinc in the presence of tetrakis(triphenylphosphine)palladium, followed by similar reactions as above gave (1-chloro-2,2-difluorovinyl)-vinyldeuteroporphyrin dimethyl esters (17 and 18).

Mobility and molecular orientation of vitamin E in liposomal membranes as determined by 19F NMR and fluorescence polarization techniques.

To elucidate the molecular orientation and mobility of alpha-tocopherol (vitamin E) in membranes, 19F nuclear magnetic resonance (NMR) was applied. In bilayer phosphatidylcholine (PC) liposomes, 19F NMR spin-spin relaxation times (T2) of 19F-labeled methyl groups on either the chromanol moiety or the isoprenoid side chain of alpha-tocopherol were significantly decreased compared to those for alpha-tocopherol in solution (CDCl3), suggesting that the vitamin E molecule exists in the lipid core of membranes and that its motional freedom is significantly restricted. The difference in T2 values between the solution state and PC liposomes was greatest for the 5a-CF3 on the chromanol moiety compared with other methyl groups on the isoprenoid side chain. Hence, it is thought that the chromanol moiety of alpha-tocopherol may fit tightly near the surface of the membrane because of its hydrophilicity. When Pr3+ was added to the liposomal suspension, the intensity of the NMR signal for 5a-CF3 changed faster than those of the other methyl groups. When the surface of the liposomal membranes was positively charged using stearyl amine, the mobility of the 5a-methyl group, expressed as the correlation time (tau c), was quite similar to that in negatively charged membranes. These results indicate that the chromanol moiety of alpha-tocopherol may orient near the membrane surface, and that the hydroxyl group may be hydrogen bonded to the ester carbonyl of PC. For further confirmation of this conclusion, time-dependent changes in the fluidity of liposomes caused by the addition of Pr3+ were measured at the surface and inner region of liposomal membranes using a fluorescence polarization technique. The fluidity increased in the surface region and decreased in the inner region of liposomes containing alpha-tocopherol. These results show that when praseodymium ions may pass through the liposomal membrane, the hydrogen bonds between alpha-tocopherol and the ester carbonyl of PC may be broken, leading to the formation of an alpha-tocopherol-Pr3+ complex, thus causing the fluidity to increase. Based on the findings obtained, mobility and molecular orientation of vitamin E in membranes are proposed.

Location and dynamics of alpha-tocopherol in model phospholipid membranes with different charges.

Studies were made on the position and dynamics of the OH-group of alpha-tocopherol in phospholipid membranes. There was no difference in the spin-lattice (T1) relaxation times at the 5a-position of alpha-tocopherol labeled with 13C- or C19F3-determined from the nuclear magnetic resonance (NMR) spectra of liposomes positively charged with stearylamine (SA) and negatively charged with dicetylphosphate (DCP). The zeta-potentials of egg yolk phosphatidylcholine (EYPC) liposomes with and without SA or DCP were not affected by incorporation of 20 mol% alpha-tocopherol, though incorporation of 10 mol% ascorbyl-palmitate decreased the zeta-potentials of EYPC and EYPC-SA liposomes. The P==O stretching band (1235 cm-1) of the phosphate group and C==O stretching band (1734 cm-1) of the acyl ester linkage in dimyristoylphosphatidylcholine (DMPC) liposomes, measured by Fourier transform-infrared (FT-IR) spectroscopy, were not changed by incorporation of alpha-tocopherol. These results suggest that no specific interaction occurred between the OH-group of alpha-tocopherol and the polar interfacial region of the bilayer. The dynamic quenching effects of n-(N-oxy-4,4'-dimethyloxazolidine-2-yl)stearic acids (n-NSs) on the intrinsic fluorescence of alpha-tocopherol were in the order 5-NS > 7-NS = 12-NS > 16-NS. Acrylamide, a water-soluble fluorescence quencher with a very low capacity to penetrate through phospholipid bilayers, had very low quenching efficiency. These results indicate that the bulk of the chromanol moiety of alpha-tocopherol is located in a position close to that occupied by the nitroxide group of 5-NS in the membranes and is poorly exposed at the membrane surface.(ABSTRACT TRUNCATED AT 250 WORDS)

Synthesis of fluorine analogues of protoporphyrin potentially useful for diagnosis and therapy of tumors.

With the aim of obtaining a porphyrin derivative useful for diagnosis and therapy of cancer, fluorine analogues of protoporphyrin, in which the vinyl group(s) were replaced by difluorovinyl group(s), were synthesized by the reaction of the formylporphyrins with sodium chlorodifluoroacetate in the presence of triphenylphosphine. Some improvements in the reported procedures for the synthesis of formylporphyrins are also described. Preliminary results of biological tests of the products showed that 8(2),8(2)-difluoroprotoporphyrin accumulates to gastric cancer more selectively than other fluorine analogues and that 3(2),3(2),8(2),8(2)-tetrafluoroprotoporphyrin is taken up by rat hepatoma cells more readily than the others.