Influence of oestradiol and tamoxifen on oestrogen receptors-alpha and -beta protein degradation and non-genomic signalling pathways in uterine and breast carcinoma cells.

Tamoxifen acts as an oestrogen antagonist in the breast reducing cell proliferation, but in the uterus as an oestrogen agonist resulting in increased cell proliferation. Tamoxifen exerts its tissue-specific effects through the oestrogen receptors (ERalpha or ERbeta). The levels and functions of the two ERs affect the response of the target tissue to oestrogen and tamoxifen. We examined the control of ER stability in breast and uterine cell lines using western blotting and RT-PCR. In MCF-7 breast-derived cells, ERalpha and ERbeta proteins were rapidly degraded via the proteasome pathway in response to oestradiol; conversely tamoxifen stabilised both receptors. In Ishikawa uterine-derived cells, oestradiol and tamoxifen stabilised ERalpha but led to degradation of ERbeta by the proteasome pathway. Further investigations showed that oestradiol induced activation of the non-genomic ERalpha/Akt signalling pathway in MCF-7 cells. We have demonstrated that the alternative Erk signalling pathway is activated in Ishikawa cells following oestradiol treatment in the absence of an active proteasome pathway and therefore increased levels of ERbeta. In conclusion, our data have demonstrated tamoxifen or oestradiol control of ER subtype stability and that non-genomic activation of transcription pathways is cell specific.

Tamoxifen: is it safe? Comparison of activation and detoxication mechanisms in rodents and in humans.

Tamoxifen, a non-steroidal antiestrogen, is the class representative of a group of drugs that include toremifene, droloxifene and idoxifene. Tamoxifen has been successfully used worldwide as adjuvant therapy in the treatment of women with breast cancer. However, such therapy results in a slightly increased risk of endometrial cancers. Lifetime exposure of rats to high doses of tamoxifen results in a high incidence of liver tumors. Tamoxifen itself is not genotoxic but is activated in the liver to alpha-hydroxytamoxifen. This is further conjugated to form the sulfate ester as the putative reactive intermediate. Studies with recombinant human CYPs show only CYP3A4 is able to catalyze the formation of alpha-hydroxytamoxifen and the irreversible binding of [(14)C]tamoxifen to DNA. CYP3A4 and CYP2D6 convert tamoxifen to N-desmethyltamoxifen. The formation 4-hydroxytamoxifen is catalyzed by CYP2D6 and at a much lower level by CYP2C19. In women, detoxication of alpha-hydroxytamoxifen via a stable glucuronide occurs at a rate in the order of 100 fold higher than in rats whereas rates of sulfation are 3 fold lower than in rats. These factors, together with the low dose of tamoxifen used therapeutically in women, indicates a minimum risk of liver cancers. Results from (32)P-postlabeling and accelerator mass spectrometry suggest that low levels of uterine DNA binding does occur but this is probably too low to play a role in uterine tumor development and it is more likely to be the estrogen agonist action of this class of drug that is the most important factor in tumor development in humans.

Comparison of the effect of oestradiol, tamoxifen and raloxifene on nerve growth factor-alpha expression in specific neonatal mouse uterine cell types using laser capture microdissection.

Oral dosing of CD-1 mice on days 2-5 after birth with tamoxifen but not raloxifene disrupts the development of the myometrium, resulting in adult uterine adenomyosis. Using laser capture microdissection and RT-PCR we have investigated nerve growth factor (NGF) and cognate receptor expression in uterine cells of 6-day-old pups that may be important in early developmental changes that give rise to adenomyosis. NGF down-regulation is known to occur during terminal myogenic differentiation. NGF was found exclusively in endometrial luminal epithelium of controls. It was up-regulated 18-fold in the luminal epithelium following dosing with tamoxifen but not raloxifene. Western blotting for NGF protein in the whole uterus showed a 25-fold increase after tamoxifen treatment. Expression of the low affinity p75 neutrophin receptor (p75(NTR)) was twofold higher in the myometrium compared with luminal epithelium or stroma. This was not altered following tamoxifen treatment. There was no detectable expression of high affinity tyrosine kinase receptor (trkA(NGFR)). This study shows luminal epithelial cells of the endometrium primarily form NGF. This suggests that NGF normally regulates the differentiation of the mesenchyme into uterine myocytes through paracrine mechanisms and that an early disturbance of this process plays a key role in the subsequent development of adenomyosis.