Abstract
This study evaluated the action of tamoxifen and estradiol on the function of isolated liver mitochondria. We observed that although tamoxifen and estradiol per se did not affect mitochondrial complexes II, III, or IV, complex I is affected, this effect being more drastic (except for state 4 of respiration) when mitochondria were coincubated with both drugs. Furthermore, using two respiratory chain inhibitors, rotenone and diphenyliodonium chloride, we identified the flavin mononucleotide site of complex I as the target of tamoxifen and/or estradiol action(s). Tamoxifen (25 microm) per se induced a significant increase in hydrogen peroxide production and state 4 of respiration. Additionally, a significant decrease in respiratory control ratio, transmembrane, and depolarization potentials were observed. Estradiol per se decreased carbonyl cyanide p-trifluoromethoxyphenylhydrazone (FCCP)-stimulated respiration, state 3 of respiration, and respiratory control ratio and increased lag phase of repolarization. With the exception of state 4 of respiration whose increase induced by tamoxifen was reversed by the presence of estradiol, the effects of tamoxifen were highly exacerbated when estradiol was present. We observed that 10 microm tamoxifen in the presence of estradiol affected mitochondria significantly by decreasing FCCP-stimulated respiration, state 3 of respiration, respiratory control ratio, and ADP depolarization and increasing the lag phase of repolarization. All of the deleterious effects induced by 25 microm tamoxifen were highly exacerbated in the presence of estradiol. Furthermore, we observed that the effects of both compounds were independent of estrogen receptors because the pure estrogen antagonist ICI 182,780 did not interfere with tamoxifen and/or estradiol detrimental effects. Altogether, our data provide a mechanistic explanation for the multiple cytotoxic effects of tamoxifen including its capacity to destroy tamoxifen-resistant breast cancer cells in the presence of estradiol. This new piece of information provides a basis for the development of new and promising anticancer therapeutic strategies.
Highlights
Estrogen by binding to the estrogen receptor (ER)2 to thereby block estradiol (E2) access [1, 3]
The fluorescence of supernatants was measured at Mitochondrial Complex I Is the Key Target of the Deleterious Effects Induced by E2 Plus TAM—In control conditions after the addition of glutamate/malate there was a slight increase in oxygen consumption caused by the activation of the mitochondrial respiratory chain (Fig. 1A)
The preincubation of mitochondria with 30 M E2 and 25 M TAM completely abolished FCCP-stimulated respiration (Fig. 1A). When these conditions were tested in mitochondrial complex II, we did not observe any statistical difference when we compared mitochondria without treatment with those incubated with E2 and/or TAM (Fig. 1B)
Summary
Materials—TAM and E2 were obtained from Sigma. DPI was obtained from Aldrich, and ICI 182,780 was obtained from TOCRIS (UK). The reactions were carried out in a chamber with magnetic stirring in 1 ml of the standard medium (130 mM sucrose, 50 mM KCl, 2.5 mM MgCl2, 2.5 mM KH2PO4, 100 M EGTA, 5 mM Hepes, pH 7.4) containing 3 M TPPϩ. This TPPϩ concentration was chosen to achieve high sensitivity in measurements and to avoid possible toxic effects on mitochondria [27, 28].
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