afimoxifene and Uterine-Neoplasms

The impact of 17beta-estradiol and antiestrogens on uterine cancer cells is poorly understood. The aim of this study was to determine the impact of 17beta-estradiol, 4-hydroxytamoxifen, raloxifene and ICI 182 780 on the cell proliferation of six uterine cancer cell lines: HeLa, HEC-1-A, KLE, RL-95-2, Ishikawa and EN-1078D. The effects of these compounds on the cell proliferation of the six uterine cancer cell lines were studied in the presence and absence of estrogens (phenol red and serum deprivation of sex steroids). In a general manner, 17beta-estradiol and 4-hydroxytamoxifen showed similarities in their effects whereas raloxifene showed a different pattern of cell proliferation (agonistic and antagonistic) and ICI 182 780 had antagonistic activity. In the presence and absence of estrogens, we observed that each cell line had diverse expression of ERalpha, ERbeta, GPR30 and REA. GPR30 mRNA expression was significantly reduced in a serum/phenol-free medium. REA mRNA expression was not influenced by the media. Results demonstrated the importance of removing phenol red and the use of deprived serum when studying uterine cancer cells in relationship with 17beta-estradiol and antiestrogens. The affinity of each compound to the binding of ERalpha and ERbeta was very similar with the exception of raloxifene that had a preference for ERalpha binding. Akt phosphorylation/activity was reduced in cells cultured in a phenol red- and steroid-free culture medium indicating that the presence of steroids in the culture media can influence the activity of this survival pathway. Our results suggest that the expression of ERalpha, ERbeta and GPR30 are influenced by sex steroids and might play a role in the response of cells to 17beta-estradiol and antiestrogens but are not the only factors involved in this process.

Topics: Antineoplastic Agents, Hormonal; Cell Line, Tumor; Cell Proliferation; Estradiol; Estrogen Antagonists; Estrogen Receptor alpha; Estrogen Receptor beta; Estrogens; Female; Fulvestrant; Gene Expression Regulation, Neoplastic; HeLa Cells; Humans; Prohibitins; Protein Binding; Raloxifene Hydrochloride; Receptors, Estrogen; Receptors, G-Protein-Coupled; Tamoxifen; Uterine Neoplasms

The effect of two anti-oestrogens, 4-OH tamoxifen and ICI 164,384, on growth and progesterone receptor (PR) concentration was investigated in the endometrial carcinoma cell line, Ishikawa. Growth stimulation in response to 4-OH tamoxifen was antagonized by ICI 164,384, the latter having no agonist effect when used as a single agent. Similarly, ICI 164,384 antagonized oestradiol-stimulated cell growth. PR was significantly increased following treatment with 4-OH tamoxifen, this response being antagonized in the presence of ICI 164,384. Oestradiol increased PR, although to a lesser extent than did 4-OH tamoxifen; the effect of oestradiol on PR was also antagonized by ICI 164,384. Used as a single agent, ICI 164,384 induced a moderate but statistically significant increase in PR, thus demonstrating partial agonist activity. This agonist property of ICI 164,384 may provide a mechanism of maintaining PR, which is down-regulated during conventional progestin therapy, without undesirable mitogenic activity.

Topics: Cell Division; Estradiol; Estrogen Antagonists; Female; Humans; Polyunsaturated Alkamides; Receptors, Progesterone; Tamoxifen; Tumor Cells, Cultured; Uterine Neoplasms

The effects of trans-4-hydroxytamoxifen (OHTam) on proliferation of cells of the Ishikawa human endometrial adenocarcinoma line were studied under serum-free, phenol red-free conditions and compared to those of estradiol. The addition of OHTam (1 microM) to basal medium (BM), consisting of equal parts of Dulbecco's modified Eagle's medium and Ham's F-12 with additional glutamine and 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid, resulted in significant increases in cell numbers relative to controls. These effects were even greater than those obtained with estradiol (10 nM-1 microM) or 1% charcoal-treated fetal bovine serum (ctFBS). Addition of 1% ctFBS to BM containing 1 microM OHTam further increased cell numbers whereas addition of estradiol (10 nM) did not do so. The stimulation of growth was positively correlated with OHTam concentrations in the range of 10 nM to 1 microM. Dissociation of estradiol and OHTam proliferative effects was observed in a variant of Ishikawa cells in which estradiol did not increase proliferation while OHTam had a strong stimulatory effect. The growth-promoting effects of OHTam were also observed in BM containing 5% or 15% ctFBS. In contrast, in parallel experiments in which BM was replaced by minimal essential medium (Eagle's) with Earle's salts, OHTam (1 microM) did not stimulate proliferation under these conditions and acted as an antiestrogen, inhibiting the proliferative effects of estradiol. These results illustrate marked effects of medium composition on proliferation and antiestrogenic actions of OHTam. Alkaline phosphatase activity was strongly stimulated by estradiol (10 nM) but only very weakly affected by OHTam (1 microM); at these concentrations, OHTam inhibited the effect of estradiol, both in serum-free BM and in minimal essential medium plus 15% ctFBS, demonstrating dissociation in its actions on proliferation and on enzymatic activity. These findings suggest that OHTam may stimulate the proliferation of particular clones of endometrial cancer cells in human tumors. They also suggest that OHTam can exert effects not mediated by the estrogen receptor system, or form OHTam-estrogen receptor agonistic complexes unlike those resulting from estradiol-estrogen receptor interactions. Clearly, Ishikawa cells provide a useful model to investigate mechanisms of action of antiestrogens.

Topics: Adenocarcinoma; Alkaline Phosphatase; Cell Division; Estradiol; Female; Fetal Blood; Humans; Tamoxifen; Tumor Cells, Cultured; Uterine Neoplasms

Effects of 4-hydroxytamoxifen, a major metabolite of tamoxifen, on the proliferation of cancer cells from human endometrial adenocarcinomas obtained by hysterectomy were investigated in primary culture. Competitive binding studies showed that 4-hydroxytamoxifen effectively binds to cytoplasmic estrogen receptors (ER) in uterine adenocarcinomas. Of 20 endometrial adenocarcinomas examined, five tumors were successfully grown in primary cell culture. The addition of 4-hydroxytamoxifen (1 nmol/l to 1 mumol/l) in a medium supplemented with estrogen-free serum resulted in a dose-dependent inhibition of the growth of cancer cells in two tumors having ER. However, 4-hydroxytamoxifen did not affect the growth in the culture system of the remaining three tumors, in which ER were absent in two tumors but were present in one. These results strongly suggest that tamoxifen has a direct growth-inhibitory effect on human endometrial adenocarcinoma possibly through ER in the tumor.

Topics: Adenocarcinoma; Binding, Competitive; Cytosol; Drug Evaluation, Preclinical; Female; Humans; Receptors, Estrogen; Tamoxifen; Tumor Cells, Cultured; Uterine Neoplasms

The effects of estradiol on DNA polymerase alpha activity were investigated in an estrogen-responsive human endometrial cancer cell line (Ishikawa) derived from a well differentiated endometrial adenocarcinoma. These cells are known to respond to estradiol by increasing progesterone receptor levels, alkaline phosphatase activity, and cell density. Four- to 5-fold increases in DNA polymerase alpha activity occurred when estradiol was added to cultures of Ishikawa cells in medium containing charcoal-treated fetal bovine serum. Maximal stimulation was achieved at 18 h during incubations with 10(-8) M estradiol, but significant effects also were found with 10(-9) and 10(-6) M. These effects were almost completely counteracted by a 100-fold excess of 4-hydroxytamoxifen. At 10(-6) M, the antiestrogen had no influence on the basal levels of DNA polymerase alpha. Medroxyprogesterone acetate (10(-6) M) was ineffective as either an enhancer of enzymatic activity or an antiestrogen when tested in combination with 10(-8) M estradiol, even in the presence of appreciable levels of specific progesterone binders. The responsiveness of the Ishikawa cells to estrogen contrasts with the lack of effects of estradiol on DNA polymerase alpha activity in another human endometrial adenocarcinoma cell line (HEC-50).

Topics: Adenocarcinoma; Cell Line; DNA Polymerase II; Enzyme Activation; Estradiol; Female; Humans; Medroxyprogesterone; Medroxyprogesterone Acetate; Tamoxifen; Uterine Neoplasms

Rabbit antibodies to cytoplasmic estrogen receptors (ER) of human breast carcinoma were utilized for investigating steroid-triggered in-vitro translocation of cytoplasmic ER to the nuclear compartment of the estrogen target cells. The immunofluorescent method (IF) previously described (S Raam et al., Eur J Cancer Clin Oncol 18: 1-12, 1982) was employed for immunohistochemical localization of ER. Four cases of normal endometrium, two cancers of the endometrium, and MCF-7 human breast cancer cells were maintained in a steroid free medium and exposed at 37 degrees C for two hours to growth medium alone (control) or to 2.5, 25 or 250 nanomoles of estradiol (E2), diethylstilbestrol (DES), or monohydroxytamoxifen (OH-TX). At the end of the incubation period the cells were processed for intracellular localization of ER. Complete traslocation of IF from the cytoplasm to the nuclear compartment was evident in all normal endometrial cells exposed to E2, DES or OH-TX for two hours. While cells from the endometrial cancer 'S', like the normal cells, translocated IF to the nucleus, cells of another cancer ('KLE') failed to translocate when exposed to E2 or OH-TX. Partial translocation was evident in 'KLE' cells exposed to DES. In MCF-7 cells grown in the absence of E2, IF was exclusively cytoplasmic. When these cells were exposed to the hormones, 50% showed a complete transfer of IF to the nucleus; in 40% a delayed response was evident; 10% failed to translocate. The results revealed the suitability of anti-ER antibodies for investigating the intracellular dynamics of ER in target cells responding to estrogens or antiestrogens.

Topics: Animals; Breast Neoplasms; Cell Line; Cell Nucleus; Cells, Cultured; Cytoplasm; Diethylstilbestrol; Endometrium; Estradiol; Estrogen Antagonists; Female; Fluorescent Antibody Technique; Humans; Immune Sera; Rabbits; Receptors, Estrogen; Tamoxifen; Uterine Neoplasms