61
Cancer Letters, 66 (1992) 61- 68 Elsevier Scientific Publishers Ireland Ltd.
Droloxifene (3-hydroxytamoxifen) has membrane antioxidant ability: potential relevance to its mechanism of therapeutic action in breast cancer Helen Wisemana, Henry
R.V.
aDepartment (UK), 95817
Cheryl Smith”, Arnstein” and Martin
of Biochemistry,
*Diuision of Pulmonary (USA)
and ‘Uniuersity
Barry Halliwellb, S. LennardC
Division of Biomolecular -
Cannona,
Sciences, King’s College London,
Critical Care Medicine,
Department
Michael
U.C.
Dauis Medical Center,
of Medicine and Pharmacology,
The
4301
Strand, London
WCZR 2LS
X Street, Sacramento,
Royal Hallamshire
Hospital,
CA
Sheffield,
510 2JF (UK) (Received
18 May 1992)
(Accepted
6 July 1992)
Summary
stabilization that could be associated in cancer cells with decreased plasma membrane jluidity. This mechanism may be related to the
Droloxijene (3-hydroxytamoxijen), is a triphenylethylene derivative recently developed for the treatment of breast cancer. Dm!~~xijene
clinically important antiprolijerative droloxijene on cancer cells.
was found to exhibit a membrane antioxidant ability in that it inhibited Fe(M)-ascorbate dependent lipid peroxidation in rat liver microsomes and ox-brain phospholipid lipo-
similarly introduced cholesterol and tamoxijen, although to a lesser extent than 17@oestradial. This inhibition of lipid’ peroxidation by droloxijene may result from a membrane Correspondence to: Helen Wiseman, Department of Biochemistry, King’s College London, Strand, London, WCZR 2LS, UK.
0304-3835/92/505.00 Printed and Published
0 1992 Elsevier Scientific Publishers in Ireland
of
Keywords: droloxifene (3-hydroxytamoxifen) ; lipid peroxidation; tamoxifen; membrane antioxidant; breast cancer; anticancer action
somes. It also inhibited microsomal lipid peroxidation induced by Fe(lll)-ADP/NADPH. Droloxijene was a better inhibitor of lipid peroxidation than tamoxijen, but was less effective than 17@-oestradiol in the two microsomal systems and in the preformed liposomal system. When introduced into oxbrain phospholipid liposomes, droloxijene inhibited Fe(N)-ascorbate induced lipid peroxidation to approximately the same extent as
action
Introduction Tamoxifen is a triphenylethylene antioestrogen drug used in the treatment and prevention of breast cancer [3,4,14,29,31]. Its 3-hydroxy derivative droloxifene has been shown to be an efficient and safe anticancer drug in phase I and early phase II clinical trials with breast cancer patients [l]. Droloxifene may have a number of advantages over tamoxifen including decreased occurrence of resistant cancer cells [1,1;,22,25,26]. In addition to acting as an oestrogen receptor antagonist tamoxifen must have other Ireland
Ltd
62
mechanisms of action, because it is effective against oestrogen receptor-negative tumours [14,28]. Oestrogen-independent actions of tamoxifen include inhibition of calmodulinmediated systems such as the plasma membrane (Ca*+ + Mg*+)-ATPase [8,27], CAMP phosphodiesterase [ 13,321 and protein kinase C, which has an important role in cellular growth regulation [5,6]. Tamoxifen is metabolized by cytochrome P-450 IIIA [21] in
humans to 4-hydroxytamoxifen and ZVdesmethyltamoxifen [24]. We have shown previously that tamoxifen and 4-hydroxytamoxifen can inhibit lipid peroxidation in phospholipid liposomes and liver microsomes [36 - 381. We have suggested that tamoxifen and 4-hydroxytamoxifen may inhibit lipid peroxidation by a membrane stabilizing action [36]. This membrane antioxidant ability led us to examine the antioxidant ability of droloxifene because of its close structural similarity to tamoxifen and 4hydroxytamoxifen (Fig. 1). Materials and Methods
TAHOXIFEN
Rl-=", R2-OCH2CH2N(CH3)2, R3=H
DROLOXIFENE
Rl=",
4-HYDROXYTAIIOXIFEN
Rl-OH, R2-OCt12Ct12N(C83)2,R3'H
&
R,
R2=OCt12CE2N(C83)2, R3=OH
CHPH
‘I
’
Rl=OH
17/)-OESTRADIOL
CH,
dP
R,
CH,
R,
\
CHOLESTEROL
Fig.1. Structures
RI-OH, R2-CH(CH3)(c"~)3cH(cH3)2
of triphenylethylene drugs, 17 fioestradiol and cholesterol. Droloxifene and tamoxifen were used as the citrate salt. Structural mimicry by tamoxifen, droloxifene and 17&oestradiol of cholesterol.
Tamoxifen (citrate salt), 17@oestradiol and cholesterol were from the Sigma Chemical Co. (Poole, UK). Droloxifene (3-hydroxytamoxifen, citrate salt), was kindly donated by Klinge Pharma (Munich, Germany). Ail other reagents were of the highest quality from the Sigma Chemical Co. (Poole, UK) or from BDH Ltd. (Dagenham, UK). Rat liver microsomes were prepared from the livers of adult male rats by standard differential-centrifugation techniques as described in [30]. Ox-brain phospholipid Iiposomes were prepared as described previously [ 16,381. Liposomes were also prepared with and without the introduction of the compounds shown in Fig. 1, as described for cholesterol [17,36,38]. Microsomal and Iiposomal lipid peroxidation in the presence of Fe(III) and ascorbate or ADP/NADPH, was measured by the formation of thiobarbituric acid-reactive substances (TBARS) as described previously [30]. The reaction mixtures (final volume of 1.0 ml) contained either microsomes (0.25 mg of microsomal protein) or liposomes (0.5 mg in 0.1 ml of phosphate buffered saline at pH 7.4); 10 mM KH2P04-KOH buffer (pH 7.4) assays and was used for microsomal phosphate buffered saline (pH 7.4) was used for liposomal L,says; 5 ~1 of ethanol or test compound dissolved in ethanol was added except in the case of liposomes already
63
containing introduced drugs or compounds. Peroxidation was started by adding aqueous solutions of Fe& (0.1 ml) and ascorbate (0.1 ml) to give a final concentration of 100 PM of each. In some experiments, microsomal lipid peroxidation was started by addition of FeCls (100 PM), ADP (1.7 mM) and NADPH (0.4 mM) to give the final concentrations stated. Freshly prepared ADP and FeCls solutions were premixed just before addition to the reaction mixture. Ascorbate or NADPH were added to start the reaction and incubations were carried out at 37OC for 20 min (unless stated otherwise). The amount of lipid peroxidation was determined by the TBA test [30]. HCl (0.5 ml, 25%, v/v) was added to each reaction mixture, followed by 0.5 ml of thiobarbituric acid solution (1%) w/v in 50 mM sodium hydroxide) and heating at 80°C for 30 min. The chromogen was extracted with 2 ml of butan-l-01 and the Ass2 of the upper (organic) layer was measured. Results
The effect of droloxifene, tamoxifen and 17& oestradiol on iron-dependent microsomal lipid peroxidation Lipid peroxidation, which can be measured by the TBA test, results from the incubation of Table 1. I&, Values for the inhibition of microsomal 17&oestradiol and cholesterol. Compound/Drug
ifene, tamoxifen and 17P-oestradiol were added to these systems at micromolar concentrations dissolved in ethanol. Figure 2 shows that in the Fe (III)-ascorbate system droloxifene was a more powerful inhibitor of lipid peroxidation than tamoxifen although less effective than 17&oestradiol. Table I shows that in the Fe(III)-ascorbate system droloxifene has an ICsO value of 13 PM compared to 17 PM for tamoxifen and 11 PM for 17/Soestradiol. Both droloxifene and 17 P-oestradiol appeared to be somewhat more powerful inhibitors in the Fe(III)-ADP/NADPH system compared to the Fe(II1) -ascorbate system, whereas for tamoxifen the converse was true (see Table I). Cholesterol dissolved in ethanol had no inhibitory effect on microsomal lipid peroxidation nor indeed did ethanol itself when used at a final concentration of 0.5% (v/v). The timecourses of peroxidation in the presence of these compounds, at their ICso concentrations (see Fig. 3) show that they all inhibited microsomal lipid peroxidation by an approximately constant percentage over the timecourse investigated. There was no evidence of a lag period followed by an acceleration of peroxidation to the control rate that is usually observed when chain-breaking antioxidants and liposomal
lipid peroxidation
by triphenylethylene
drugs,
Systems Microsomal FeWasc.
Liposomal FeIII-ADP/NADPH
(PM)
17 fi-estradiol Tamoxifen Droloxifene Cholesterol
rat liver microsomes with Fe(III)-ascorbate [34] or Fe(III)-ADP/NADPH [7] at pH 7.4. Drolox-
11 17 13 N.R.
9 19 11 N.R.
Added to preformed (/MI
Introduced into during preparation (moles/mole) (mMI
11 62 38 N.R.
0.04 6.1 7.2 7.2
0.0044 0.63 0.75 0.75
N.R., not reached i.e. inhibition by the drug or compound does not reach 50%. Asc., ascorbate. Droloxifene and tamoxifen were used as the citrate salt. Values are deduced from the graphs shown in Figs. 2, 4 and 6 in which each point represents the meen f S.D. of 3- 10, 3-8 and 3-9, respectively, separate assays.
64
90
10 0 0
5
10
15
20
25
30
Drug concentration (pi) 0
Fi2. 2.
Concentration-dependent inhibition of microsomal lipid peroxidation induced by Fe& and ascorbate. m 17&oestradiol, (0) tamoxifen, (A) droloxifene. Droloxifene and tamoxifen were used as the citrate salt. Results are mean f S.D., n = 3- 10 tests.
are added to peroxidizing microsomes. Droloxifene, tamoxifen and 17@-oestradiol did not interfere with the TBA test: no inhibition was observed when the compounds were added to peroxidizing microsomes along with the TBA reagents instead of at the beginning of the incubations. The effect of droloxifene, tamoxifen and 17/3oestradiol on iron-dependent liposomal lipid peroxidation ox-brain from formed Liposomes phospholipids have been shown to provide a model membrane (lipid bilayer) system, which in the presence of Fe(III)-ascorbate at pH 7.4 is rapidly peroxidised [9] as measured by the TBA test. Droloxifene, tamoxifen and 17/3oestradiol, dissolved in ethanol, were added to preformed ox-brain phospholipid liposomes. Figure 4 shows that droloxifene is again a more
5
10
15
20
Time (min)
Fig. 3.
Time course of microsomal lipid peroxidation induced by FeCI, and ascorbate: the effect of test compounds added at their I&, concentrations. ( L ) control (ethanol only added); m 13 PM 17&oestradiol, (0) 17 PM tamoxifen, (A) 14 pM droloxifene. Droloxifene and tamoxifen were used as the citrate salt. Results shown are the means of duplicate determinations. Concentrations quoted are the final concentrations in the reaction mixtures.
powerful inhibitor than tamoxifen although much less potent than 17fl-oestradiol. This is also reflected in the IC& values of the compounds (see Table I), which were 38 PM for droloxifene compared to 62 PM for tamoxifen and only 11 PM for 17&oestradiol. The timecourses for liposomal lipid peroxidation (see Fig. 5) again suggest that these triphenylethylene drugs and 17fl-oestradiol are not antioxidants. chain-breaking acting as Cholesterol was again not an inhibitor of lipid peroxidation in this system.
65
0
o
5 Drug
10
15
concentration
20
25
5
10
Time
(min)
15
20
30
(p-l)
Fig. 4. Concentration-dependent inhibition of liposomal lipid peroxidation induced by FeQ and ascorbate. (Wj 17@-oestradiol, (0) tamoxifen and (A) droloxifene. Droloxifene and tamoxifen were used as the citrate salt. The drugs were dissolved in ethanol and added to preformed liposomes. Results are mean f S.D., n = 3-8 tests.
The effect of droloxifene, tamoxifen and 17& oestradiol introduced into liposomes during their preparation on iron-dependent liposomal lipid peroxidation The introduction of cholesterol into ox-brain phospholipid liposomes during their preparation inhibits lipid peroxidation apparently by membrane stabilization [17,36]. We, therefore, studied the effect of similarly introducing cholesterol, tamoxifen, droloxifene and 17PThe compound/phospholipid oestradiol. ratios were expressed in molar terms using an average relative molecular mass for phospholipids based on the reported phospholipid composition [ 171. The data in Fig. 6 expressed as molar ratios (phospholipid at 5 mg/ml) show that droloxifene introduced into liposomes over the concentration range
Fig. 5. Time course of liposomal lipid peroxidation induced by FeCls and ascorbate: the effect of test compounds added at their IC, concentrations. ( h ) control (ethanol only added), R 11 FM 17&oestradioi, (0) 62 PM tamoxifen, (A) 38 PM droloxifene. Droloxifene and tamoxifen were used as the citrate salt. Results shown are the means of duplicate determinations. Concentrations quoted are the final concentrations in the reaction mixtures.
0.01- 8 mg/ml inhibited lipid peroxidation to approximately the same extent as similarly introduced cholesterol (0.01-8 mg/ml) and to a slightly lesser extent than tamoxifen (0.01 - 8 mg/ml). Droloxifene was, however, a much less effective inhibitor of lipid peroxidation than 17&oestradiol (0.005 - 4 mg/ml). This is reflected in the IC& values (Table I) of droloxifene (7.2 mM) , tamoxifen (6.1 mM) and 17&oestradiol (0.04 mM) compared to cholesterol (7.2 mM) . Discussion Droloxifene was a more effective inhibitor of lipid peroxidation than tamoxifen but was less effective than 17@-oestradiol in the microsomal and preformed liposomal systems. This is in
66 100
90
10 0 10-3
10-2
10-l
compound:phospholipid
10"
10'
boles/mole)
Fig. 6. Inhibition of lipid peroxidation by the compounds, (A) cholesterol, (4 17/3-oestradiol, (0) tamox-
ifen, (A) droloxifene each introduced into liposomes during their preparation. Droloxifene and tamoxifen were used as the citrate salt. Results are mean f S.D., R = 3 - 9 tests.
contrast to 4-hydroxytamoxifen, which was found previously to be a more powerful antioxidant than either tamoxifen or 17p-
oestradiol in these systems [36 - 381. The results presented here for droloxifene and 17& oestradiol and previously for 4-hydroxytamoxifen [36-381 suggest that these drugs, which all possess a hydroxyl group (and tamoxifen which does not) do not exert their antioxidant action via a chain-breaking mechanism. This is because the peroxidation time-courses do not exhibit the pronounced lag-period expected for chain-breaking antioxidants [ 181. Both droloxifene and tamoxifen, when inphospholipid into ox-brain troduced liposomes, appear to exert a membrane stabilizing action against lipid peroxidation comparable to that shown by cholesterol. This
may occur via an interaction between the hydrophobic rings of cholesterol and the saturated, mono-unsaturated and polyunsaturated fatty acid side-chains of phospholipids [17,36,38]. In this system the hydroxyl group of droloxifene (R3 = OH; see Fig. 1) did not increase the ability of this drug to inhibit lipid peroxidation compared with tamoxifen. This is in contrast to the observations in the preformed liposomal system (see above). The RBA (relative binding affinity) for the oestrogen receptor of 4-hydroxytamoxifen is similar to that of 17@-oestradiol and is 100-fold higher than that of tamoxifen [14], while that of droloxifene is IO-fold higher than that of tamoxifen [26]. This is indicative of the importance of the ring position of the hydroxyl group on the tamoxifen molecule in relation to structural mimicry of the sterols cholesterol and 17@-oestradiol, which we have previously examined using molecularcomputer modelling techniques [35]. We suggest that droloxifene also displays a membranestabilizing action, allowing it to act as an antioxidant. The membrane-stabilizing action of droloxifene might be expected to cause a decrease in membrane fluidity (as demonstrated recently for human breast cancer cells treated with tamoxifen [lo]) that would antagonize cell division in cancer cells, as already proposed for tamoxifen [35,36,38]. Metastatic tumour cells have a higher plasma membrane fluidity than non-metastatic cells [33] and thus decreased membrane fluidity could restore contact-inhibition between such cells by increasing the rigidity of the cells at celljunction contacts. This may counteract the metastasis of invasive epithelial malignancies of the breast, which is aided by the generation of stromelysin-3 in the surrounding stromal cells [2]. Moreover, the action of adenylate cyclase has been shown to be inhibited by decreased membrane fluidity [20], which would result in lowered CAMP levels shown to inhibit the growth of some cancer cells [12]. The concept of the cell membrane as a target for cancer therapy is poorly developed [15] and we have extended this approach through
67
consideration of the effect of decreased membrane fluidity. The advantages of using droloxifene rather than tamoxifen, which include decreased oestrogen agonism, faster clearance and thus decreased opportunity for drug resistant cancer cells to arise [1,11,22,25,26] compared to tamoxifen usage [19,23] should lead to its increased use in prevention as well as treatment of breast cancer. Therefore, assessment of the membrane antioxidant action of droloxifene reported here in relation to its anticancer action is timely and of considerable potential importance in identifying useful new derivatives of tamoxifen and other triphenylethylene-based drugs for “hormone-therapy” approaches to breast cancer treatment and prevention. Acknowledgements
The award of a Leverhulme Emeritus Fellowship to H.R.V. Arnstein is gratefully acknowledged. We thank the Charities Aid Foundation, the British Heart Foundation and the Cancer Research Campaign for financial support and the S.E.R.C for a studentship to C. Smith. References
6
7 8
9
10
11
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13
14
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Ahlemann, L.M., Staab, H.J., Loser, R., Seibel, K. and Huber, H.J. (1988) lnhibition of growth of human cancer by intermittent exposure to the antioestrogen droloxifene. Tumour Diagn. Ther., 9, 41-46. Basset, P., Bellocq, J.P., Wolf, C., Stall, I., Hutin. P., Limacher, J.M., Podhajer, O.L., Chenard, M.P., Rio, M.C. and Chambon, P. (1990) A novel metalloproteinase gene specifically expressed in stromal ceils of breast carcinomas. Nature, 348, 699- 704. Baum, M., Ziv, Y. and Colletta, A. (1991) Can we prevent breast cancer. Br. J. Cancer, 64, 205-207. Baum, M., Ziv, Y. and Colletta, A. (1991) Prospects for the chemoprevention of breast cancer. Br. Med. Bull., 47, 493 - 503. Bignon, E., Pons, M., Dorc, J.C., Gilbert, J., Ojasoo, T., Miquel. J.F., Raynaud, J.P. (1991) Influence of diphenylethylene and triphenylethylene estrogen/antiestrogen structure on the mechanisms of protein kinase C inhibition and activation as revealed by a multivariate analysis. Biochem. Pharmacol., 42, 1373- 1384.
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Bignon, E., Pons, M., Gilbert, J. and Nishizuka, Y. (1990) Multiple mechanisms of protein kinase C inhibition by triphenylacrylnitrile antioestrogens. FEBS Lett , 27 1, 54-58. Buege, J.A. and Aust, SD. (1978) Microsomal lipid peroxidation. Methods Ennymol., 30, 303 - 310. Celeste, M., Lopes, F., Graca, M., Vale, P. and Carvalho. binding of tamoxifen to A.P. (1990) Ca *+-dependent calmodulin isolated from bovine brain. Cancer Res.. 50, 2753 - 2758. Chatterjee, S.N. and Agarwal, S. (1988) Liposomes as membrane model for study of lipid peroxidation. Free Rad. Biol. Med., 4, 51- 72. Clarke, R., van den Berg, H.W. and Murphy, R.F. (1990) Reduction of the membrane fluidity of human breast cancer cells by tamoxifen and 17P-estradiol, J. Natl. Cancer Inst., 82, 1702- 1705. Dietel, M., Loser, R., Rohlke, P., Jonat, W., Niendorf, A., Gerding, D., Kohr, A., Holzel, F. and Arps. H. (1989) Effect of continuous vs. intermittent application of 3-OHtamoxifen or tamoxifen on the proliferation of the human breast cancer cell line MCF-7Ml. Cancer Res. Clin. Oncoj., 115, 36-40. Dumont, J.E., Jauniaux, J.C. and Roger, P.P. (1989) The cyclic AMP-mediated stimulation of cell proliferation Trends Biochem. Sci., 14, 67 - 71. Fanidi, A., Pageaux, J.F.. Courtson. C., Fayard, J.M., Laugier, C. (1991) Growth inhibition and the regulation of cyclic AMP phosphodiesterase by the triphenylethylene anti-estrogen tamoxifen in the quail oviduct. Biol. Cell, 72, 181- 188. Fun, B.J.A. and Jordan, V.C. (1984) The pharmacology and clinical uses of tamoxifen. Pharmacol. Therapeut., 25, 127 - 205. Grunicke, H. (1991) The cell membrane as a target for cancer chemotherapy. Eur. J. Cancer, 27, 281- 284. Gutteridge, J.M.C. (1977) The measurement of malondialdehyde in peroxidized ox-brain phospholipid liposomes. Anal. Biochem., 82, 76-82. Gutteridge, J.M.C. (1978) The membrane effects of vitamin E, cholesterol and their acetates on peroxidative susceptibility. Res. Commun. Chem. Pathol. Pharmacol., 22, 563 - 572. Halliwell, B. (1990) HOW to characterize a biological antioxidant. Free Radical Res. Commun., 9, l-32. Katzenellenbogen, B.S. (1991) Antioestrogen resistance - mechanism by which breast cancer cells undermine the effectivness of endocrine therapy. J. Nat]. Cancer Inst., 83, 1434 - 1435. Houslay, M.D. (1985) Regulation of adenylate cyclase (EC.4.6.1.1) activity by its lipid environment. Proc. Nutr. Sot., 44, 157 - 165. Jacolot. F.. Simon, I., Dreano, Y., Beaune, P.. Riche, C., Berthou, F. (1991) Identification of the cytochrome P-450 IllA family as the enzymes involved in the N-demethylation of tamoxifen in human liver microsomes. Biochem. Pharmacol., 41, 1911- 1919.
68 22
23
24
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30
Kawamura, I., Miiota, T., Mukumoto, S., Manda, K., Nakamura, T., Kubota, H., Matsumoto, S., Nishigata, F., Shimomura, K., Mori, J., Masugi, T. and Shibayama, F. (1989) Antioestrogenic and antitumour effects of droloxifene in experimental breast cancer. Anneim. Forsch./Drug Res., 39, 889 - 893. Kellen, J.A., George, E. and Ling, V. (1991) Decreased P-glycoprotein in a tamoxifen tolerant breast carcinoma model. Anticancer Res., 11, 1243- 1244. Lien, E.A., Solheim, E. and Ueland, P.M. (1991) Distribution of tamoxifen and its metabolites in rat and human tissues during steady-state treatment. Cancer Res., 51, 4837 - 4844. Loser, R., Seibel, K. and Huber, H.J. (1988) Pharmacological activities of droloxifene isomers. Anticancer Res., 8, 1271- 1274. Loser, R., Seibel, K. Liehn, H.D. and Staab, H.J. (1986) Pharmacology and toxicology of the antioestrogen droloxifene. Contr. Oncol., 23, 64-72. Malva, J.O., Lopes, M.C.F., Vale, M.G.P. and Carvalho, A.P. (1990) Action of antioestrogens on the (Ca*+ + Mg*+)-ATPase and Na+/Ca+ exchange of brain cortex membranes. Biochem. Pharmacol., 40, 1877 - 1884. Murphy, L.C. and Sutherland, R.L. (1985) Differential effects of tamoxifen and analogs with nonbasic side chains on cell proliferation in vitro. Endocrinol., 116, 1071- 1078. Nayfield, S.C., Karp, J.E., Ford, L.G., Dorr, F.A. and Kramer, B.S. (1991) Potential role of tamoxifen in prevention of breast cancer. J. Natl. Cancer Inst., 83, 1450 - 1459. Quinlan, G.J., Halliwell, B., Moorhouse, C.P. and Gutteridge, J.M.C. (1988) Action of lead (II) and aluminium (III) ions on iron-stimulated lipid peroxidation in liposomes,
31 32
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erythrocytes and rat liver microsomal fractions. Biochim., Biophys. Acta, 962, 196-200. Richardson, D.N. (1988) The history of Nolvadex. Drug Design Delivery, 3, 1- 14. Rowlands, M.G., Parr, LB., McCague, R., Jarman, M. and Goddard, P.M. (1990) Variation of the inhibition of calmodulin dependent cyclic AMP phosphodiesterase amongst analogues of tamoxifen; correlations with cytotoxicity. Biochem. Pharmacol., 40, 283-289. Taraboletti, G., Perin, L. and Bottazzi, B., (1989) Membrane fluidity affects tumour-cell motility, invasion and lung-colonizing potential. Int. J. Cancer, 44, 707 - 713. Wills, E.D. (1969) Lipid peroxide formation in microsomes. The role of non-haem iron. Biochem. J., 113, 325-332. Wiseman, H., Cannon, M., Amstein, H.R.V. and Barlow, D.J. (1992), The structural mimicry of membrane sterols by tamoxifen: evidence from cholesterol coefficients and molecular-modelling for its action as a membrane antioxidant and as an anticancer agent. Biochim. Biophys. Acta, 1138, 197 - 202. Wiseman, H., Cannon, M. Arnstein, H.R.V. and Halliwell, B. (1990) Mechanism of inhibition of lipid peroxidation by tamoxifen and 4-hydroxytamoxifen introduced into liposomes: Similarity to cholesterol and ergosterol. Fed. Eur. Biochem. Sot. Lett., 274, 107 - 110. Wiseman, H., Laughton, M.J., Arnstein, H.R.V., Cannon, M. and Halliwell, B. (1990) The antioxidant action of tamoxifen and its metabolites. Inhibition of lipid peroxidation. Fed. Eur. Biochem. Sot. Lett., 263, 192- 194. Wiseman, H., Smith, C., Amstein, H.R.V., Halliwell, B. and Cannon, M. (1991) The antioxidant action of ketoconazole and related azoles: comparison with tamoxifen and cholesterol. Chem.-Biol. Interact., 79, 229- 243.
Cancer Letters, 66 (1992) 61- 68 Elsevier Scientific Publishers Ireland Ltd.
Droloxifene (3-hydroxytamoxifen) has membrane antioxidant ability: potential relevance to its mechanism of therapeutic action in breast cancer Helen Wisemana, Henry
R.V.
aDepartment (UK), 95817
Cheryl Smith”, Arnstein” and Martin
of Biochemistry,
*Diuision of Pulmonary (USA)
and ‘Uniuersity
Barry Halliwellb, S. LennardC
Division of Biomolecular -
Cannona,
Sciences, King’s College London,
Critical Care Medicine,
Department
Michael
U.C.
Dauis Medical Center,
of Medicine and Pharmacology,
The
4301
Strand, London
WCZR 2LS
X Street, Sacramento,
Royal Hallamshire
Hospital,
CA
Sheffield,
510 2JF (UK) (Received
18 May 1992)
(Accepted
6 July 1992)
Summary
stabilization that could be associated in cancer cells with decreased plasma membrane jluidity. This mechanism may be related to the
Droloxijene (3-hydroxytamoxijen), is a triphenylethylene derivative recently developed for the treatment of breast cancer. Dm!~~xijene
clinically important antiprolijerative droloxijene on cancer cells.
was found to exhibit a membrane antioxidant ability in that it inhibited Fe(M)-ascorbate dependent lipid peroxidation in rat liver microsomes and ox-brain phospholipid lipo-
similarly introduced cholesterol and tamoxijen, although to a lesser extent than 17@oestradial. This inhibition of lipid’ peroxidation by droloxijene may result from a membrane Correspondence to: Helen Wiseman, Department of Biochemistry, King’s College London, Strand, London, WCZR 2LS, UK.
0304-3835/92/505.00 Printed and Published
0 1992 Elsevier Scientific Publishers in Ireland
of
Keywords: droloxifene (3-hydroxytamoxifen) ; lipid peroxidation; tamoxifen; membrane antioxidant; breast cancer; anticancer action
somes. It also inhibited microsomal lipid peroxidation induced by Fe(lll)-ADP/NADPH. Droloxijene was a better inhibitor of lipid peroxidation than tamoxijen, but was less effective than 17@-oestradiol in the two microsomal systems and in the preformed liposomal system. When introduced into oxbrain phospholipid liposomes, droloxijene inhibited Fe(N)-ascorbate induced lipid peroxidation to approximately the same extent as
action
Introduction Tamoxifen is a triphenylethylene antioestrogen drug used in the treatment and prevention of breast cancer [3,4,14,29,31]. Its 3-hydroxy derivative droloxifene has been shown to be an efficient and safe anticancer drug in phase I and early phase II clinical trials with breast cancer patients [l]. Droloxifene may have a number of advantages over tamoxifen including decreased occurrence of resistant cancer cells [1,1;,22,25,26]. In addition to acting as an oestrogen receptor antagonist tamoxifen must have other Ireland
Ltd
62
mechanisms of action, because it is effective against oestrogen receptor-negative tumours [14,28]. Oestrogen-independent actions of tamoxifen include inhibition of calmodulinmediated systems such as the plasma membrane (Ca*+ + Mg*+)-ATPase [8,27], CAMP phosphodiesterase [ 13,321 and protein kinase C, which has an important role in cellular growth regulation [5,6]. Tamoxifen is metabolized by cytochrome P-450 IIIA [21] in
humans to 4-hydroxytamoxifen and ZVdesmethyltamoxifen [24]. We have shown previously that tamoxifen and 4-hydroxytamoxifen can inhibit lipid peroxidation in phospholipid liposomes and liver microsomes [36 - 381. We have suggested that tamoxifen and 4-hydroxytamoxifen may inhibit lipid peroxidation by a membrane stabilizing action [36]. This membrane antioxidant ability led us to examine the antioxidant ability of droloxifene because of its close structural similarity to tamoxifen and 4hydroxytamoxifen (Fig. 1). Materials and Methods
TAHOXIFEN
Rl-=", R2-OCH2CH2N(CH3)2, R3=H
DROLOXIFENE
Rl=",
4-HYDROXYTAIIOXIFEN
Rl-OH, R2-OCt12Ct12N(C83)2,R3'H
&
R,
R2=OCt12CE2N(C83)2, R3=OH
CHPH
‘I
’
Rl=OH
17/)-OESTRADIOL
CH,
dP
R,
CH,
R,
\
CHOLESTEROL
Fig.1. Structures
RI-OH, R2-CH(CH3)(c"~)3cH(cH3)2
of triphenylethylene drugs, 17 fioestradiol and cholesterol. Droloxifene and tamoxifen were used as the citrate salt. Structural mimicry by tamoxifen, droloxifene and 17&oestradiol of cholesterol.
Tamoxifen (citrate salt), 17@oestradiol and cholesterol were from the Sigma Chemical Co. (Poole, UK). Droloxifene (3-hydroxytamoxifen, citrate salt), was kindly donated by Klinge Pharma (Munich, Germany). Ail other reagents were of the highest quality from the Sigma Chemical Co. (Poole, UK) or from BDH Ltd. (Dagenham, UK). Rat liver microsomes were prepared from the livers of adult male rats by standard differential-centrifugation techniques as described in [30]. Ox-brain phospholipid Iiposomes were prepared as described previously [ 16,381. Liposomes were also prepared with and without the introduction of the compounds shown in Fig. 1, as described for cholesterol [17,36,38]. Microsomal and Iiposomal lipid peroxidation in the presence of Fe(III) and ascorbate or ADP/NADPH, was measured by the formation of thiobarbituric acid-reactive substances (TBARS) as described previously [30]. The reaction mixtures (final volume of 1.0 ml) contained either microsomes (0.25 mg of microsomal protein) or liposomes (0.5 mg in 0.1 ml of phosphate buffered saline at pH 7.4); 10 mM KH2P04-KOH buffer (pH 7.4) assays and was used for microsomal phosphate buffered saline (pH 7.4) was used for liposomal L,says; 5 ~1 of ethanol or test compound dissolved in ethanol was added except in the case of liposomes already
63
containing introduced drugs or compounds. Peroxidation was started by adding aqueous solutions of Fe& (0.1 ml) and ascorbate (0.1 ml) to give a final concentration of 100 PM of each. In some experiments, microsomal lipid peroxidation was started by addition of FeCls (100 PM), ADP (1.7 mM) and NADPH (0.4 mM) to give the final concentrations stated. Freshly prepared ADP and FeCls solutions were premixed just before addition to the reaction mixture. Ascorbate or NADPH were added to start the reaction and incubations were carried out at 37OC for 20 min (unless stated otherwise). The amount of lipid peroxidation was determined by the TBA test [30]. HCl (0.5 ml, 25%, v/v) was added to each reaction mixture, followed by 0.5 ml of thiobarbituric acid solution (1%) w/v in 50 mM sodium hydroxide) and heating at 80°C for 30 min. The chromogen was extracted with 2 ml of butan-l-01 and the Ass2 of the upper (organic) layer was measured. Results
The effect of droloxifene, tamoxifen and 17& oestradiol on iron-dependent microsomal lipid peroxidation Lipid peroxidation, which can be measured by the TBA test, results from the incubation of Table 1. I&, Values for the inhibition of microsomal 17&oestradiol and cholesterol. Compound/Drug
ifene, tamoxifen and 17P-oestradiol were added to these systems at micromolar concentrations dissolved in ethanol. Figure 2 shows that in the Fe (III)-ascorbate system droloxifene was a more powerful inhibitor of lipid peroxidation than tamoxifen although less effective than 17&oestradiol. Table I shows that in the Fe(III)-ascorbate system droloxifene has an ICsO value of 13 PM compared to 17 PM for tamoxifen and 11 PM for 17/Soestradiol. Both droloxifene and 17 P-oestradiol appeared to be somewhat more powerful inhibitors in the Fe(III)-ADP/NADPH system compared to the Fe(II1) -ascorbate system, whereas for tamoxifen the converse was true (see Table I). Cholesterol dissolved in ethanol had no inhibitory effect on microsomal lipid peroxidation nor indeed did ethanol itself when used at a final concentration of 0.5% (v/v). The timecourses of peroxidation in the presence of these compounds, at their ICso concentrations (see Fig. 3) show that they all inhibited microsomal lipid peroxidation by an approximately constant percentage over the timecourse investigated. There was no evidence of a lag period followed by an acceleration of peroxidation to the control rate that is usually observed when chain-breaking antioxidants and liposomal
lipid peroxidation
by triphenylethylene
drugs,
Systems Microsomal FeWasc.
Liposomal FeIII-ADP/NADPH
(PM)
17 fi-estradiol Tamoxifen Droloxifene Cholesterol
rat liver microsomes with Fe(III)-ascorbate [34] or Fe(III)-ADP/NADPH [7] at pH 7.4. Drolox-
11 17 13 N.R.
9 19 11 N.R.
Added to preformed (/MI
Introduced into during preparation (moles/mole) (mMI
11 62 38 N.R.
0.04 6.1 7.2 7.2
0.0044 0.63 0.75 0.75
N.R., not reached i.e. inhibition by the drug or compound does not reach 50%. Asc., ascorbate. Droloxifene and tamoxifen were used as the citrate salt. Values are deduced from the graphs shown in Figs. 2, 4 and 6 in which each point represents the meen f S.D. of 3- 10, 3-8 and 3-9, respectively, separate assays.
64
90
10 0 0
5
10
15
20
25
30
Drug concentration (pi) 0
Fi2. 2.
Concentration-dependent inhibition of microsomal lipid peroxidation induced by Fe& and ascorbate. m 17&oestradiol, (0) tamoxifen, (A) droloxifene. Droloxifene and tamoxifen were used as the citrate salt. Results are mean f S.D., n = 3- 10 tests.
are added to peroxidizing microsomes. Droloxifene, tamoxifen and 17@-oestradiol did not interfere with the TBA test: no inhibition was observed when the compounds were added to peroxidizing microsomes along with the TBA reagents instead of at the beginning of the incubations. The effect of droloxifene, tamoxifen and 17/3oestradiol on iron-dependent liposomal lipid peroxidation ox-brain from formed Liposomes phospholipids have been shown to provide a model membrane (lipid bilayer) system, which in the presence of Fe(III)-ascorbate at pH 7.4 is rapidly peroxidised [9] as measured by the TBA test. Droloxifene, tamoxifen and 17/3oestradiol, dissolved in ethanol, were added to preformed ox-brain phospholipid liposomes. Figure 4 shows that droloxifene is again a more
5
10
15
20
Time (min)
Fig. 3.
Time course of microsomal lipid peroxidation induced by FeCI, and ascorbate: the effect of test compounds added at their I&, concentrations. ( L ) control (ethanol only added); m 13 PM 17&oestradiol, (0) 17 PM tamoxifen, (A) 14 pM droloxifene. Droloxifene and tamoxifen were used as the citrate salt. Results shown are the means of duplicate determinations. Concentrations quoted are the final concentrations in the reaction mixtures.
powerful inhibitor than tamoxifen although much less potent than 17fl-oestradiol. This is also reflected in the IC& values of the compounds (see Table I), which were 38 PM for droloxifene compared to 62 PM for tamoxifen and only 11 PM for 17&oestradiol. The timecourses for liposomal lipid peroxidation (see Fig. 5) again suggest that these triphenylethylene drugs and 17fl-oestradiol are not antioxidants. chain-breaking acting as Cholesterol was again not an inhibitor of lipid peroxidation in this system.
65
0
o
5 Drug
10
15
concentration
20
25
5
10
Time
(min)
15
20
30
(p-l)
Fig. 4. Concentration-dependent inhibition of liposomal lipid peroxidation induced by FeQ and ascorbate. (Wj 17@-oestradiol, (0) tamoxifen and (A) droloxifene. Droloxifene and tamoxifen were used as the citrate salt. The drugs were dissolved in ethanol and added to preformed liposomes. Results are mean f S.D., n = 3-8 tests.
The effect of droloxifene, tamoxifen and 17& oestradiol introduced into liposomes during their preparation on iron-dependent liposomal lipid peroxidation The introduction of cholesterol into ox-brain phospholipid liposomes during their preparation inhibits lipid peroxidation apparently by membrane stabilization [17,36]. We, therefore, studied the effect of similarly introducing cholesterol, tamoxifen, droloxifene and 17PThe compound/phospholipid oestradiol. ratios were expressed in molar terms using an average relative molecular mass for phospholipids based on the reported phospholipid composition [ 171. The data in Fig. 6 expressed as molar ratios (phospholipid at 5 mg/ml) show that droloxifene introduced into liposomes over the concentration range
Fig. 5. Time course of liposomal lipid peroxidation induced by FeCls and ascorbate: the effect of test compounds added at their IC, concentrations. ( h ) control (ethanol only added), R 11 FM 17&oestradioi, (0) 62 PM tamoxifen, (A) 38 PM droloxifene. Droloxifene and tamoxifen were used as the citrate salt. Results shown are the means of duplicate determinations. Concentrations quoted are the final concentrations in the reaction mixtures.
0.01- 8 mg/ml inhibited lipid peroxidation to approximately the same extent as similarly introduced cholesterol (0.01-8 mg/ml) and to a slightly lesser extent than tamoxifen (0.01 - 8 mg/ml). Droloxifene was, however, a much less effective inhibitor of lipid peroxidation than 17&oestradiol (0.005 - 4 mg/ml). This is reflected in the IC& values (Table I) of droloxifene (7.2 mM) , tamoxifen (6.1 mM) and 17&oestradiol (0.04 mM) compared to cholesterol (7.2 mM) . Discussion Droloxifene was a more effective inhibitor of lipid peroxidation than tamoxifen but was less effective than 17@-oestradiol in the microsomal and preformed liposomal systems. This is in
66 100
90
10 0 10-3
10-2
10-l
compound:phospholipid
10"
10'
boles/mole)
Fig. 6. Inhibition of lipid peroxidation by the compounds, (A) cholesterol, (4 17/3-oestradiol, (0) tamox-
ifen, (A) droloxifene each introduced into liposomes during their preparation. Droloxifene and tamoxifen were used as the citrate salt. Results are mean f S.D., R = 3 - 9 tests.
contrast to 4-hydroxytamoxifen, which was found previously to be a more powerful antioxidant than either tamoxifen or 17p-
oestradiol in these systems [36 - 381. The results presented here for droloxifene and 17& oestradiol and previously for 4-hydroxytamoxifen [36-381 suggest that these drugs, which all possess a hydroxyl group (and tamoxifen which does not) do not exert their antioxidant action via a chain-breaking mechanism. This is because the peroxidation time-courses do not exhibit the pronounced lag-period expected for chain-breaking antioxidants [ 181. Both droloxifene and tamoxifen, when inphospholipid into ox-brain troduced liposomes, appear to exert a membrane stabilizing action against lipid peroxidation comparable to that shown by cholesterol. This
may occur via an interaction between the hydrophobic rings of cholesterol and the saturated, mono-unsaturated and polyunsaturated fatty acid side-chains of phospholipids [17,36,38]. In this system the hydroxyl group of droloxifene (R3 = OH; see Fig. 1) did not increase the ability of this drug to inhibit lipid peroxidation compared with tamoxifen. This is in contrast to the observations in the preformed liposomal system (see above). The RBA (relative binding affinity) for the oestrogen receptor of 4-hydroxytamoxifen is similar to that of 17@-oestradiol and is 100-fold higher than that of tamoxifen [14], while that of droloxifene is IO-fold higher than that of tamoxifen [26]. This is indicative of the importance of the ring position of the hydroxyl group on the tamoxifen molecule in relation to structural mimicry of the sterols cholesterol and 17@-oestradiol, which we have previously examined using molecularcomputer modelling techniques [35]. We suggest that droloxifene also displays a membranestabilizing action, allowing it to act as an antioxidant. The membrane-stabilizing action of droloxifene might be expected to cause a decrease in membrane fluidity (as demonstrated recently for human breast cancer cells treated with tamoxifen [lo]) that would antagonize cell division in cancer cells, as already proposed for tamoxifen [35,36,38]. Metastatic tumour cells have a higher plasma membrane fluidity than non-metastatic cells [33] and thus decreased membrane fluidity could restore contact-inhibition between such cells by increasing the rigidity of the cells at celljunction contacts. This may counteract the metastasis of invasive epithelial malignancies of the breast, which is aided by the generation of stromelysin-3 in the surrounding stromal cells [2]. Moreover, the action of adenylate cyclase has been shown to be inhibited by decreased membrane fluidity [20], which would result in lowered CAMP levels shown to inhibit the growth of some cancer cells [12]. The concept of the cell membrane as a target for cancer therapy is poorly developed [15] and we have extended this approach through
67
consideration of the effect of decreased membrane fluidity. The advantages of using droloxifene rather than tamoxifen, which include decreased oestrogen agonism, faster clearance and thus decreased opportunity for drug resistant cancer cells to arise [1,11,22,25,26] compared to tamoxifen usage [19,23] should lead to its increased use in prevention as well as treatment of breast cancer. Therefore, assessment of the membrane antioxidant action of droloxifene reported here in relation to its anticancer action is timely and of considerable potential importance in identifying useful new derivatives of tamoxifen and other triphenylethylene-based drugs for “hormone-therapy” approaches to breast cancer treatment and prevention. Acknowledgements
The award of a Leverhulme Emeritus Fellowship to H.R.V. Arnstein is gratefully acknowledged. We thank the Charities Aid Foundation, the British Heart Foundation and the Cancer Research Campaign for financial support and the S.E.R.C for a studentship to C. Smith. References
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