Michael Cannon

The antioxidant action of tamoxifen and its metabolites. Inhibition of lipid peroxidation.

The anti‐oestrogen drug tamoxifen is an inhibitor of lipid peroxidation in rat liver microsomes and in phospholipid liposomes. Its cis isomer and N‐desmethyl form are weaker inhibitors, but 4‐hydroxytamoxifen is much more powerful. It is possible that the antioxidant property of tamoxifen might contribute to its biological actions.

Ribosomes in thiostrepton-resistant mutants of Bacillus megaterium lacking a single 50 S subunit protein.

Abstract A protein required for the binding of thiostrepton to ribosomes of Bacillus megaterium has been purified and further characterized by immunological techniques. This protein, which does not bind the drug off the ribosome, is serologically-homologous to Escherichia coli ribosomal protein L11 and is designated BM-L11. Ribosomes from certain thiostrepton-resistant mutants of B. megaterium appear to be totally devoid of protein BM-L11 as judged by modified immunoelectrophoresis. Such ribosomes are significantly less sensitive than those from wild-type organisms to the action of thiostrepton in vitro but retain substantial protein synthetic activity. Re-addition of protein BM-L11 to ribosomes from the mutants restores them to wild-type levels of activity and thiostrepton sensitivity. Thus ribosomal protein BM-L11 is involved not only in binding thiostrepton but also in determining the thiostrepton phenotype.

The structural mimicry of membrane sterols by tamoxifen: evidence from cholesterol coefficients and molecular-modelling for its action as a membrane anti-oxidant and an anti-cancer agent

The anti-cancer drug tamoxifen is a potent inhibitor of lipid peroxidation induced by Fe(III)-ascorbate in ox-brain phospholipid liposomes. Similar anti-oxidant effects, but with varying potencies, are also shown by 4-hydroxy-tamoxifen, cholesterol, ergosterol and 17-beta-oestradiol. We now describe a computer-graphic fitting technique that demonstrates a structural similarity between the five compounds. In addition, we have quantified the differences (relative to cholesterol) between the anti-oxidant activities of the compounds in terms of a novel expression referred to here as the cholesterol coefficient (Cc) Finally, we discuss how the inhibitory effect of tamoxifen on lipid peroxidation may result from a membrane stabilization that is associated with a decrease in membrane fluidity. This action may be related to the anti-proliferative effect exerted by tamoxifen on cancer and fungal cells.

Mechanism of inhibition of lipid peroxidation by tamoxifen and 4-hydroxytamoxifen introduced into liposomes. Similarity to cholesterol and ergosterol.

The anticancer drug tamoxifen when introduced into phospholipid liposomes during their preparation inhibited Fe(III)‐ascorbate induced lipid peroxidation to a greater extent than similarly introduced cholesterol. Ergosterol was equipotent with tamoxifen, but much less effective than 4‐hydroxytamoxifen. Possible mechanisms underlying these effects are discussed in relation to structural mimicry of the sterols by these triphenylethylene drugs as membrane stabilizers against lipid peroxidation.

Enhancement by tamoxifen of the membrane antioxidant action of the yeast membrane sterol ergosterol: relevance to the antiyeast and anticancer action of tamoxifen.

The anticancer drug tamoxifen inhibits lipid peroxidation in ox-brain phospholipid liposomes, and is a good antiyeast agent, with clinical potential. We now report that the ergosterol-containing lipid fraction derived from yeast microsomal membranes (and the ergosterol separated from it) inhibited lipid peroxidation when introduced into ox-brain phospholipid liposomes. Inhibition of lipid peroxidation by the lipid fraction was greatly enhanced when yeast cell growth was inhibited with tamoxifen prior to lipid extraction. The ability of tamoxifen to enhance the membrane antioxidant ability of ergosterol is expressed in terms of a tamoxifen enhancement coefficient. Enhancement by tamoxifen of the membrane antioxidant action of ergosterol is discussed in relation to the antifungal and anticancer actions of tamoxifen.

Inhibition at the initiation level of eukaryotic protein synthesis by T-2 toxin

The 12,13-epoxytrichothecenes inhibit protein synthesis in eukaryotic organisms [l] and can block peptidyl transferase activity as assayed by the puromycin fragment reaction [2]. However, on the basis of experiments in vivo they have been divided into two subgroups [3]. It has been proposed that compounds in one subgroup (I-type inhibitors) which include T-2 toxin, act at some stage of the initiation process. Compounds in the second subgroup (E-type inhibitors) include trichodermin and seem to inhibit elongation or perhaps more specifically termination [41. The 12,13-epoxytrichothecenes are closely related chemically [5] and it has been suggested thai they have the same site of action on ribosomes [6]. It remains, therefore, to establish why the two subgroups apparently act at different stages of the protein synthetic cycle. Recent work with trichodermin has strongly suggested that this compound acts as a general inhibitor of the peptidyl transferase catalytic centre [6] and it is tempting to assume that T-2 toxin also inhibits this same enzymic centre but, for some reason, only at the initiation step of protein synthesis. In this present work we have utilized the reticulocyte cell-free system to study the effect of T-2 toxin on the initiation process. We have found that T-2 toxin can inhibit the reaction which can take place between puromycin and met-tRNAmft bound to the 80 S ribosome.

Modes of action of erythromycin and thiostrepton as inhibitors of protein synthesis

During protein synthesis in cell-free extracts of Escherichia coli peptidyl-tRNA is located on one of two ribosomal binding sites [ 1 ] . The nascent peptide is transfered from tRNA bound at the P site on to amino acyl-tRNA bound at an adjacent A site [2]. This reaction requires an enzyme, peptidyl transferase, which is an integral part of the 50 S subunit [3] . Before the peptide chain can elongate further, peptidyl-tRNA bound at site A must be translocated to site P to allow a further amino acyl-tRNA to attach at site A. Translocation requires GTP hydrolysis and ‘G’ factor [4]. Puromycin reacts with peptidyl-tRNA bound to site P to release peptidyl-puromycin [S, 61 . This reaction requires only the correct ionic environment and is catalysed by peptidyl transferase [7]. The puromycin reaction can thus be used to study the formation of an individual peptide bond. Translocation can also be detected using puromycin since more peptide moves to site P. Finally, the reaction assays the distribution of peptidyl-tRNA between sites A and P in a given ribosome population. We have utilised the puromycin reaction to elucidate the mechanisms of action of two antibiotics thiostrepton and elythromycin. Both have been proposed to inhibit translocation [8,9] . Studies on washed ribosomes have suggested that thiostrepton, but not erythromycin, may inhibit translocation. After inhibition, by thiostrepton or erythromycin, of crude extracts actively synthesising protein, thiostrepton causes peptidyl-tRNA to accumulate in site P. Erythromycin apparently causes peptidyl-tRNA to ac-

Tamoxifen inhibits lipid peroxidation in cardiac microsomes. Comparison with liver microsomes and potential relevance to the cardiovascular benefits associated with cancer prevention and treatment by tamoxifen.

Tamoxifen and 4-hydroxytamoxifen were both good inhibitors of iron-dependent lipid peroxidation in rat cardiac microsomes. Tamoxifen was also a good inhibitor of lipid peroxidation in liposomes prepared from the phospholipid obtained from rat liver microsomes. In a modified rat liver microsomal system containing a sufficiently low amount of peroxidizable phospholipid to make it comparable with the rat cardiac microsomal system, tamoxifen and 4-hydroxytamoxifen were of similar effectiveness as in the cardiac system. Tamoxifen is known to lower serum cholesterol levels, and the findings reported here indicate that the drug might also protect heart cell membranes against peroxidative damage. Potential cardioprotective and antiatherosclerotic benefits of tamoxifen are discussed in relation to the drugs use in cancer prevention and treatment.

Methylation of proteins in 40 S ribosomal subunits from Saccharomyces cerevisiae

Some of the ribosomal proteins of Escherichiu coli are methylated [l-7] and since the biological significance of protein methylation is not currently understood the phenomenon provides an important field of study. Post-translational methylation of ribosomal proteins may, for example, play a vital role in the assembly of the active ribosome and its subsequent functioning in protein synthesis. Comb et al. [8] observed that ribosomal proteins from the aquatic fungus Blastocladiella emersonii could be methylated in vitro and methylation of ribosomal proteins has now been demonstrated in HeLa cells [9] although the proteins concerned were not identified. Terhorst et al. [ 1,2] have observed that proteins L7 and L12, in E. coli 50 S ribosomal subunits, are methylated and Alix and Hayes [4] have now claimed that in the same organism the ribosomal protein Ll 1 is heavily methylated and contains three methyl groups in a single trimethyllysine residue and an equal number in another unidentified amino acid. Similar results have been reported by Chang et al. [5] and these same authors have shown that at least six and possibly ten proteins from the 50 S ribosomal subunit of E. coli are methylated both in vitro [6] and in vivo [5,7]. In the present investigation we have studied the methylation, in vivo, of proteins from the 60 S ribo-

Droloxifene (3-hydroxytamoxifen) has membrane antioxidant ability: potential relevance to its mechanism of therapeutic action in breast cancer

Droloxifene (3-hydroxytamoxifen), is a triphenylethylene derivative recently developed for the treatment of breast cancer. Droloxifene was found to exhibit a membrane antioxidant ability in that it inhibited Fe(III)-ascorbate dependent lipid peroxidation in rat liver microsomes and ox-brain phospholipid liposomes. It also inhibited microsomal lipid peroxidation induced by Fe(III)-ADP/NADPH. Droloxifene was a better inhibitor of lipid peroxidation than tamoxifen, but was less effective than 17 beta-oestradiol in the two microsomal systems and in the preformed liposomal system. When introduced into ox-brain phospholipid liposomes, droloxifene inhibited Fe(III)-ascorbate induced lipid peroxidation to approximately the same extent as similarly introduced cholesterol and tamoxifen, although to a lesser extent than 17 beta-oestradiol. This inhibition of lipid peroxidation by droloxifene may result from a membrane stabilization that could be associated in cancer cells with decreased plasma membrane fluidity. This mechanism may be related to the clinically important antiproliferative action of droloxifene on cancer cells.